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1. Direct ELISA (antigen-coated plate; screening antibody)

2. Indirect ELISA (antigen-coated plate; screening antigen/antibody

3. Sandwich ELISA (antibody-coated plate screening antigen)

4. Competitive ELISA (screening antibody)

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ADVANTAGES and DISADVANTAGES of ENZYME LINKED IMMUNOASSAY.

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ADVANTAGES and DISADVANTAGES of ENZYME LINKED IMMUNOASSAY.

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The discovery of antigen-antibody reactions paved way to the advancement of serological testing

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The discovery of antigen-antibody reactions paved way to the advancement of serological testing

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Quick Overview of how Immunoprecipitation works

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Quick Overview of how Immunoprecipitation works

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Immunoprecipitation (IP) is the small-scale affinity purification of antigens using a specific antibody that is immobilized to a solid support such as magnetic particles or agarose resin.

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"Correlation Of ELISA Method With Three Other Automated Serological Tests For The Detection Of Anti-SARS-Cov-2 Antibodies"

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"Correlation Of ELISA Method With Three Other Automated Serological Tests For The Detection Of Anti-SARS-Cov-2 Antibodies"

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Nguyen, Nguyen N., Manohar B. Mutnal, Richard R. Gomez, Huy N. Pham, Lam T. Nguyen, William Koss, and Arundhati Rao et al. 2020. "Correlation Of ELISA Method With Three Other Automated Serological Tests For The Detection Of Anti-SARS-Cov-2 Antibodies". PLOS ONE 15 (10): e0240076. doi:10.1371/journal.pone.0240076.

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Nguyen N. Nguyen,

Manohar B. Mutnal,

Richard R. Gomez,

Huy N. Pham,

Lam T. Nguyen,

William Koss,

Arundhati Rao,

Alejandro C. Arroliga,

Liping Wang,

Dapeng Wang

Subject Areas

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Abstract

Public health emergency of SARS-CoV-2 has facilitated diagnostic testing as a related medical countermeasure against COVID-19 outbreak. Numerous serologic antibody tests have become available through an expedited federal emergency use only process. This paper highlights the analytical characteristic of an ELISA based assay by AnshLabs and three random access immunoassay (RAIA) by DiaSorin, Roche, and Abbott that have been approved for emergency use authorization (EUA), at a tertiary academic center in a low disease-prevalence area. The AnshLabs gave higher estimates of sero-prevalence, over the three RAIA methods. For positive results, AnshLabs had 93.3% and 100% agreement with DiaSorin or Abbott and Roche respectively. For negative results, AnshLabs had 74.3% and 78.3% agreement with DiaSorin and Roche or Abbott respectively. All discrepant samples that were positive by AnshLabs and negative by RAIA tested positive by all-in-one step SARS-CoV-2 Total (COV2T) assay performed on the automated Siemens Advia Centaur XPT analyzer. None of these methods, however, are useful in early diagnosis of SARS-CoV-2.

Citation: Nguyen NN, Mutnal MB, Gomez RR, Pham HN, Nguyen LT, Koss W, et al. (2020) Correlation of ELISA method with three other automated serological tests for the detection of anti-SARS-CoV-2 antibodies. PLoS ONE 15(10): e0240076. https://doi.org/10.1371/journal.pone.0240076

Editor: Padmapriya P. Banada, Rutgers University, UNITED STATES

Received: June 26, 2020; Accepted: September 20, 2020; Published: October 6, 2020

Copyright: © 2020 Nguyen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and Supporting Information files.

Funding: Baylor Scott and White Health provided support for the study in the form of salaries for all authors, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section.

Competing interests: Baylor Scott and White Health provided support for the study in the form of salaries for all authors. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare. All authors have no other potential competing financial, non-financial, professional, or personal interests.

Introduction

The SARS-CoV-2 virus outbreak that began in late 2019 in Wuhan, has a mortality rate of approximately 6.1% worldwide [ 1- 3]. Diagnostic testing is necessary for identifying and isolating infected individuals to limit spread of disease. Molecular testing such as reverse-transcriptase polymerase chain reaction (rtPCR) detects active infection; and serology testing helps identify those who were previously infected (including asymptomatic infections) and have recovered [ 4, 5]. Nucleic acid detection using rtPCR has become the confirmation test, due to its 99% specificity and 60-90% sensitivity within 7 days of exposure [ 6] but is faced with numerous supply challenges [ 7]. The United States Food and Drug Administration (FDA) issued an Emergency Use Approval (EUA) authorization for antibody testing as complementary to rtPCR, leading to an explosion of new antibody methods, including rapid diagnostic test (RDT), enzyme-linked immunosorbent assay (ELISA), virus neutralization assay (VNA), and chemiluminescent immunoassay (CLIA). These methods offer a range of sensitivities; the RDT provides results in less than 30 min for the presence or absence of antibodies against the virus in a whole blood specimen but has the lowest sensitivity, ELISA and CLIA can quantify antibodies to the virus in about 2-5 hours and 0.5-1 hour respectively in either serum or plasma; while VNA can quantify presence of active antibodies that are able to inhibit virus growth ex vivo, but requires 3-5 days [ 8, 9]. The best clinical utility of antibody testing for efficient diagnosis at tertiary medical centers remains unclear for screening asymptomatic patients and is being considered for identifying patients with adaptive immune responses for convalescent plasma donor program, or for treating re-positive cases [ 10]. Additionally the relative performance of many of these assays remains unclear.

We evaluated the performance of COVID-19 serology testing on three random access immunoassay analyzers (RAIA) that are typically found in clinical laboratory across US-Architect i2000 (Abbott Laboratories, Chicago IL), Cobas e601 (Roche Laboratories, Indianapolis, IN), and Liaison XL (DiaSorin, Stillwater, MN)-comparing their performance to an ELISA based assay (AnshLabs, Webster, TX) and rtPCR test (Luminex Corporation, Austin, TX). The ELISA microtiter plate-based immunoassay, was automated on Dynex DSX instrument ( Dynex Technologies, Chantilly, VA, USA) for testing IgG and IgM in serum or plasma.

Materials and methods

Specimen selection

This project used randomly selected 167 left-over convenience human serum specimens that were de-identified and stored at -20°C. The inclusion criteria included i) residual sample volume of > 1.5 mL and ii) documented rtPCR result for SARS-CoV-2. All samples were from patients who were either hospitalized with a confirmed COVID-19 diagnosis, seen in the Emergency Department with symptoms for COVID-19, or were screened for COVID-19 before an elective surgery procedure. Fifteen of the 167 samples were from patients that tested positive by rtPCR with a confirmed COVID-19 clinical diagnosis. These samples were drawn >13 days after rtPCR testing. One hundred and fifty-two serum samples were from patients who tested negative by rtPCR, 134 of these were collected on same day as rtPCR testing. For the remaining 18 samples, the interval between rtPCR and sample collection ranged from 1-48 days. To avoid degradation, the specimens were tested by four methodologies within 12-20 h of each other.

Instrumentation and analysis

Table 1 summarizes the characteristics of the four serologic assays we investigated.

The AnshLabs SARS-CoV2 IgG assay is based on the ELISA technique that measures antibodies to spike and nucleocapsid proteins. It is for in-vitro diagnostic use only and is performed on the Dynex automated analyzer. Serum samples are diluted in a culture tube and transferred to the microtitration wells coated with purified SARS-CoV-2 recombinant antigen. They are incubated for 30 min at 37°C along with calibrators. The wells are washed and treated with the anti-human IgG antibodies conjugate labeled with peroxidase. After a second incubation and washing step, the wells are incubated with the substrate tetramethylbenzidine (TMB) chromogen solution to induce color change. An acidic stopping solution is added and the degree of enzymatic turnover of the substrate is determined by wavelength absorbance measurement, with 450 nm as the primary filter and 630 nm as the reference filter. The intensity of color change corresponds to arbitrary units of antibody-antigen complex concentration present in the specimen. The analyzer calculates antibody concentration in arbitrary concentration units (AU/mL). Samples with AU/mL of >12, 10-12, and <10 are considered positive, indeterminate and negative for IgG respectively. It is the only test that uses a three-point calibration curve. The sensitivity and specificity are 95.0% and 98.3% respectively [ 11].

The Abbott SARS-CoV-2 IgG assay was run on the Abbott Architect i2000SR analyzer that measures IgG antibodies to the nucleocapsid protein. The automated, two-step immunoassay uses chemiluminescent microparticle immunoassay (CMIA) technology for qualitative detection of IgG antibodies in human serum. The sample, SARS-CoV-2 antigen-coated paramagnetic microparticles, and diluent are combined and incubated. The antibodies bind to the antigen-coated microparticles. The mixture is washed and anti-human IgG acridinium-labeled conjugate is added. Following incubation, the pre-trigger is added. The resulting chemiluminescent reaction is measured as a relative light unit (RLU). The presence or absence of IgG antibodies is determined by dividing the sample RLU by the stored calibrator RLU to find the IgG assay index (S/C), with a positive cutoff of ≥1.4. The sensitivity and specificity are 100% and 99.63% respectively at ≥ 14 days post onset of symptoms [ 12].

The LIAISON SARS-Cov-2 S1/S2 IgG is a chemiluminescent immunoassay (CLIA) for detection of anti-S1 and anti-S2 spike glycoprotein specific to SARS-CoV-2 in human serum or plasma on the DiaSorin XL analyzer (Stillwater, MN). Specimen, calibrator, control, coated magnetic particles and diluent are incubated in reaction cuvettes. The antibodies bind to the solid phase through the recombinant S1 and S2 antigens. A second incubation links recombinant S1 and S2 antigens to an isoluminol-antibody conjugate. The starter reagents are then added, and a flash chemiluminescence reaction induced. The light signal, and hence the amount of isoluminol-antibody conjugate, is measured by a photomultiplier and result converted to arbitrary concentration, AU/mL. Samples with AU/mL of ≥15 are considered positive for IgG antibodies. The sensitivity and specificity are 90-97% and 98% respectively ≥ 14 days post onset of symptoms [ 13].

The Elecsys Anti-SARS-CoV-2 assay is performed on the Roche cobas e601 analyzer for total antibodies specific for IgG, IgM and IgA which target nucleocapsid protein, in human serum or plasma. A 20uL sample and biotinylated SARS-CoV-2 specific recombinant antigen labeled with ruthenium bind in the first incubation. In the second incubation, streptavidin-coated solid phase microparticles are added to help bind the complex to the solid phase via interaction between biotin and streptavidin. The reaction mixture is aspirated into cells where microparticles are captured on the surface of electrode, and the unbound substances are washed out with ProCell solution. The ruthenylated-labeled antigen mediates detection via electrochemiluminescence, which is measured by a photomultiplier tube. Results are calculated by software, comparing the electrochemiluminescence signal of the sample to the cutoff value of the calibration as a cutoff index (COI). Samples with COI ≥1.0 are considered reactive or positive for anti-SARS-COV-2 antibodies. The sensitivity and specificity are 65.5-100% and 99.81% respectively [ 14]. The result by all methodologies are reported qualitatively as positive or negative for SARS-COV2.

Precision and specificity analysis.

The precision studies were carried out by testing pooled positive and negative patient specimens for 5 consecutive days in duplicate. No discrepant results were noted, i.e. all positive and negative were consistent.

Dilution studies.

In order to rule out non-specific binding, samples that tested positive by ELISA assay were diluted using sample diluent provided in the AnshLabs assay kit. We made and reran samples for a 1:2, 1:3 and 1:4 dilution, and calculated percent recovery.

Third party adjudication studies.

All ELISA and RAIA discordant samples were evaluated against the FDA emergency used approved all-in-one step SARS-CoV-2 Total (COV2T) assay performed on the automated Siemens Advia Centaur XPT analyzer in a reference laboratory.

Statistical analysis.

All test results were collated using a Microsoft Excel (Microsoft, Redmond, WA) spreadsheet. Statistical comparison was done using qualitative analysis on EP Evaluator Quality Assurance Simplified software (South Burlington, Vermont).

Results

The specificities of the validated in-house AnshLabs SARS-CoV-2-IgG and IgM are listed in Table 2. The cross reactivity to anti-influenza B IgG (5 samples), anti-respiratory syncytial virus IgG (5 samples), anti-nuclear antibodies (5 samples), rheumatoid factors (5 samples), anti-influenza A IgG (5 samples), anti-HCV IgG (5 samples), anti-HBV IgG (5 samples), anti-Haemophilus influenza IgG (5 samples) and anti-HIV (5 samples) was determined by testing 45 patient samples obtained before the pandemic and were positive for these analytes. No cross-reactivity was noted for either SARS-CoV-2-IgG or IgM. The clinical sensitivity and specificity using rtPCR results as the gold standard were found to be 86.7% and 91.2% respectively. All samples used for the sensitivity and specificity evaluation were collected from symptomatic patients, either hospitalized inpatients or treated in Emergency Department. The interval between rtPCR confirmation and serology testing ranged from 2-12 days.

Table 3 shows the percent agreement between ELISA and RAIA results for samples that were confirmed positive for SARS-CoV-2 by rtPCR. These samples were collected from symptomatic patients post rtPCR confirmation. ELISA assay correlated best with Total Antibody assay on Roche Elecsys e601 analyzer. This could possibly be attributed to the measurement of IgG antibodies directed towards multiple antigenic proteins (nucleocapsid & spike) by ELISA or measurement of total antibodies (IgG, IgM, and IgA) on Roche Elecsys e601 analyzer.

Table 4 shows the agreement between ELISA and RAIA for samples from patients that tested negative for SARS-CoV-2 by rtPCR. The ELISA assay showed an agreement ranging from 74.3-78.3% with different RAIA methodologies: 34, 1, 7, and 5 patients that had tested negative by rtPCR tested positive for antibodies by ELISA, Architect i2000, Liaison XL and Elecsys e601 methodology respectively. All 34 samples that tested positive by ELISA were confirmed positive by Siemens all-in-one step SARS-CoV-2 Total (COV2T) assay on the Siemens Advia Centaur XPT analyzer performed at a nationally acclaimed reference laboratory. This illustrates a higher rate of sero-prevalence is observed by ELISA versus RAIA.

The agreement of ELISA and RAIA results with rtPCR samples tested >13 days previously is shown in Table 5. Nine out of 10 samples tested positive by both rtPCR and all serologic methods. One sample tested negative by rtPCR, but tested positive by all serologic methods. Three RAIA methodologies showed high correlation with nucleic acid test for patient samples that tested negative by rtPCR, with agreements ranging from 95.4-99.3%. The ELISA assay on the other hand showed an agreement of 77.6% for these rtPCR negative samples.

The non-specific binding dilution experiment of the AnshLabs assay showed five samples with various concentration levels of IgG serially diluted to 1:2, 1:4: 1:8 and 1:16. All samples gave a consistent dilution pattern and expected 90-100% recovery of neat sample in AU/mL units ( Fig 1).

AU/mL = Arbitrary unit per milliliter, which is provided by the manufacturer.

Discussion

The RAIA methods had 100% and 90% correlation with ELISA and rtPCR respectively for samples collected >13 days post rtPCR confirmation. There were no significant differences among the methods which tested for IgG targeted to one or both nucleocapsid and spike proteins, or tested for total antibodies.

ELISA detected higher sero-prevalence in rtPCR negative samples than the RAIA methods. This may be due to i) higher analytical sensitivity or a lower cutoff by ELISA, which triggered more positive results; ii) cross reactivity to other coronavirus; iii) non-specific binding of other antibodies, for example autoimmune antibodies or deposition of detection antibody on the microtiter well which led to increased absorbance causing false positives. ELISA assays are generally known for low detection limits in sub ng/mL to low pg/mL because of their increased incubation time thereby allowing antigen-antibody to reach reaction equilibrium and extra washing steps [ 15, 16]. The Dynex DSX analyzer used for ELISA assay provided optimization flexibility and automation, which is not available on RAIA due to throughput constraint. Non-specific deposition of other antibodies in patient samples or detection antibody was ruled out by dilution studies for ELISA. Recovery of 90-110% ruled out non-specific binding as a possible cause for false positives ( Fig 1). The difference in results for positive and negative samples by RAIA methods may also be due to a higher threshold for positivity.

We believe that higher rate of positivity observed for ELISA i.e. 34 versus 1 by Architect, 7 by Liaison XL and 5 by Elecsys e601, is the net effect of extra washing and longer incubation times used by ELISA or a higher S/C cutoff set in RAIA assays. These are not false positives as claimed in other studies [ 17, 18] but are true positives not picked up by RAIA. This inadvertently decreases identification of infected patients 5-10 days post infection. The recently released all-in-one step SARS-CoV-2 Total (COV2T) assay performed on the automated Siemens RAIA-Advia Centaur XPT analyzer may have resolved some of these issues and it correlated well with our in-house ELISA assay by detecting all 34 samples that were missed by other RAIA as positives.

Project limitations

Our quality assurance project has some notable limitations. At this stage of the disease, true clinical sensitivity and specificity for different methodologies is difficult to determine because of our limited understanding of the disease process and kinetics. Secondly, our assumption that ELISA has better limits of detection is based on circumstantial evidence, as certified standards quantifying limits of detection on different platforms are not available. Third, the cutoffs provided by manufacturers were relied on which may not have undergone extensive validation. Establishing laboratory specific cut-off is akin to establishing reference ranges, which is highly dependent on prevalence of disease in local population.

Conclusion

All of the assays we investigated would work well for epidemiological sero-prevalence studies. Among rtPCR negative patients, ELISA gave higher estimates of sero-prevalence in our dataset and would probably do so in population-based epidemiological surveys using serological testing. RAIA methods could however offer other advantages over ELISA which includes i) faster turnaround time; ii) random access to allow immediate testing; iii) longer calibration stability, obviating the need to perform daily calibration as required by ELISA; iv) the ability to perform other immunoassay testing concurrently; and v) higher test throughput and walk away capabilities. However in conclusion, no serological method tested has sensitivity and specificity greater than or equal to 99% for one to 5 days post exposure, limiting their use in early diagnosis.

Acknowledgments

The authors would like to thank Ms. Briget Da Graca for editorial revision. We also acknowledge Ms. Laura Gonazales and her team from Health Texas Provider Network (Dallas, TX) for blinded testing of discrepant samples between ELISA and RAIA methods using Siemens Centaur total antibody assay.

References

1. 4. Okba NMA, Muller MA, Li W, Wang C, GeurtsvanKessel CH, Corman VM, et al. SARS-CoV-2 specific antibody responses in COVID-19 patients. medRxiv. 2020:2020.03.18.20038059.

2. 7. European Centre for Disease Prevention and Control. Novel coronavirus disease 2019 (COVID-19) pandemic: increased transmission in the EU/EEA and the UK-sixth update- 12 March 2020. Stockholm: ECDC, 2020.

3. 8. Lassaunière R, Frische A, Harboe ZB, Nielsen AC, Fomsgaard A, Krogfelt KA, et al. Evaluation of nine commercial SARS-CoV-2 immunoassays. medRxiv. 2020:2020.04.09.20056325.

4. 9. Wu F, Wang A, Liu M, Wang Q, Chen J, Xia S, et al. Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 recovered patient cohort and their implications. medRxiv. 2020:2020.03.30.20047365.

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Correlation of ELISA method with three other automated serological tests for the detection of anti-SARS-CoV-2 antibodies

# data[].attributes.attachment_link.description
Public health emergency of SARS-CoV-2 has facilitated diagnostic testing as a related medical countermeasure against COVID-19 outbreak. Numerous serologic antibody tests have become available through an expedited federal emergency use only process.

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An ELISA-Based Method For Detection Of Rabies Virus Nucleoprotein-Specific Antibodies In Human Antemortem Samples

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An ELISA-Based Method For Detection Of Rabies Virus Nucleoprotein-Specific Antibodies In Human Antemortem Samples

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Realegeno, Susan, Michael Niezgoda, Pamela A. Yager, Amrita Kumar, Laboni Hoque, Lillian Orciari, Suryaprakash Sambhara, Victoria A. Olson, and Panayampalli Subbian Satheshkumar. 2018. "An ELISA-Based Method For Detection Of Rabies Virus Nucleoprotein-Specific Antibodies In Human Antemortem Samples". PLOS ONE 13 (11): e0207009. doi:10.1371/journal.pone.0207009.

# data[].attributes.attachment_link.readable_text
Susan Realegeno,

Michael Niezgoda,

Pamela A. Yager,

Amrita Kumar,

Laboni Hoque,

Lillian Orciari,

Suryaprakash Sambhara,

Victoria A. Olson,

Panayampalli Subbian Satheshkumar

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Abstract

Rabies is a fatal encephalitic disease in humans and animals caused by lyssaviruses, most commonly rabies virus (RABV). Human antemortem diagnosis of rabies is a complex process involving multiple sample types and tests for the detection of antibodies, antigen (protein), and nucleic acids (genomic RNA). Serological diagnosis of human rabies includes the detection of either neutralizing or binding antibodies in the cerebrospinal fluid (CSF) or serum samples from unimmunized individuals without prior rabies vaccination or passive immunization with purified immunoglobulins. While neutralizing antibodies are targeted against the surface-expressed glycoprotein (G protein), binding antibodies to viral antigens are predominantly against the nucleoprotein (N protein), although there can be antibodies against all RABV-expressed proteins. To determine N protein-specific antibody responses in the CSF and serum during RABV infection, we developed an enzyme-linked immunosorbent assay (ELISA) with purified recombinant N protein expressed in E. coli. N protein-specific immunoglobulin (Ig) subtypes IgG and IgM were detected in the CSF or serum of previously diagnosed human rabies cases. In addition, anti-N protein seroconversion was demonstrated over the course of illness in individual rabies cases. We compared the N protein ELISA results to those of an indirect fluorescent antibody (IFA) test, the current binding antibody assay used in diagnosis, and show that our ELISA is consistent with the IFA test. Sensitivity and specificity of the N protein ELISA ranged from 78.38-100% and 75.76-96.77% with respect to the IFA results. Our data provide evidence for the use of an N protein ELISA as an additional option for the detection of RABV-specific IgG or IgM antibodies in human CSF or serum specimens.

Citation: Realegeno S, Niezgoda M, Yager PA, Kumar A, Hoque L, Orciari L, et al. (2018) An ELISA-based method for detection of rabies virus nucleoprotein-specific antibodies in human antemortem samples. PLoS ONE 13(11): e0207009. https://doi.org/10.1371/journal.pone.0207009

Editor: Charles E. Rupprecht, Wistar Institute, UNITED STATES

Received: May 2, 2018; Accepted: October 23, 2018; Published: November 7, 2018

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Rabies is a viral disease that results in acute encephalitis in both human and animals. It is caused by neurotropic lyssaviruses, such as RABV, a single-stranded negative sense RNA virus belonging to the Lyssavirus genus and Rhabdoviridae family [ 1]. The genome of RABV encodes five proteins, including the N protein, which along with phosphoprotein (P) and RNA-dependent RNA polymerase (L) encapsulates the genomic RNA to form the ribonucleoprotein complex (RNC). The core RNC is surrounded by the viral envelope consisting of G and matrix (M) proteins. Immunogenicity to RABV has been reported primarily against G, N, or the RNC, which is mostly comprised of N protein [ 2- 5]. Previous studies have demonstrated that immunization with either G or N proteins expressing recombinant vectors can offer protection against RABV infection in animal models [ 6- 8]. In addition, N protein can enhance the adaptive immune responses, including neutralizing antibody activity [ 3, 9, 10]. Thus, G and N proteins are both ideal target antigens for measuring antibody responses following RABV vaccination or infection.

Detection of antibodies against RABV is an important indicator of immune and exposure status. Presence of anti-RABV antibodies in the CSF or in the serum of an unvaccinated individual is considered a positive human antemortem diagnosis for rabies [ 11], provided no passive immunization with intravenous immune globulin containing anti-RABV antibodies has occurred [ 12]. The two methods currently used for determination of anti-RABV antibodies are the rapid fluorescent focus inhibition test (RFFIT) [ 13] and the IFA test [ 14]. The RFFIT is a functional assay in which the ability of antibodies to inhibit RABV infection is determined. Since G protein is responsible for RABV entry and fusion, RFFIT detects only G protein-specific antibodies [ 15, 16]. The IFA test, on the other hand, provides information on binding antibodies (including non-neutralizing) which can be targeted against any of the RABV encoded proteins, although it is demonstrated to be predominantly against the G and N proteins [ 17]. The IFA has the advantage of being able to differentiate Ig subclasses, such as IgG or IgM, the latter being an indicator for recent or primary infection. Both of these methods require the use of infectious virus and a trained technician to visualize and recognize RABV-specific staining patterns. Alternatively, several ELISA based investigative antibody detection methods have been developed. While most ELISA methods use the G protein as the preferred target antigen [ 18- 22], few have used the N protein [ 23- 26]. However, the utility of these ELISA tests in human rabies antemortem diagnosis has not been demonstrated.

The RABV N protein is the most transcriptionally abundant protein during infection [ 27- 29] and is typically identified in infected tissues in the form of inclusions [ 30]. Because of its high expression level, antigen detection diagnostic tests, like the direct fluorescent antibody (DFA) test and direct rapid immunohistochemical test (DRIT), utilize polyclonal or monoclonal antibodies (mAbs) against RABV N protein [ 31- 33]. In addition, antigenic typing also involves the use of epitope-specific mAbs against the N protein to identify RABV-specific variants from infected specimens [ 34, 35]. Similarly, the intracytoplasmic inclusion staining patterns seen in the IFA test, which utilizes RABV-infected cells for detection of RABV-specific binding antibodies, resembles that of N protein [ 29, 36]. Therefore, N protein is an ideal target antigen for use in a binding antibody detection assay.

Here, we developed an N protein ELISA and investigated the immune response to RABV in specimens collected from previously diagnosed human rabies cases. These results were compared with the IFA test, the current binding assay used in the antemortem diagnosis of rabies. Similar to the IFA test, the N protein ELISA was able to detect both IgG and IgM subclasses in CSF and serum samples from individuals positively diagnosed with RABV infection. We propose that the N protein ELISA can be an additional tool for antibody detection of RABV-specific antibodies in human CSF and serum samples. The advantages of the ELISA are the ability to use recombinant protein and a colorimetric detection method without the need for infected samples or a fluorescence microscope, features that can be suitable for resource limited environments.

Materials and methods

RABV N protein expression and purification

Purified N protein was obtained from ARVYS Proteins, Inc involving the following protocol. In brief, the N gene sequence for RABV CVS (Challenge virus standard) 11 strain was codon optimized and chemically synthesized. It was cloned into pET100D expression vector (Thermo Fisher Scientific) with an N-terminal Histidine tag for efficient purification. The plasmid was transformed into E. coli DE3 Star expression cells, culture was subjected to cell lysis and the soluble fraction was purified using Ni-affinity chromatography. Purity of the N protein sample was evaluated in a 4-12% Bis-Tris polyacrylamide gel (Thermo Fisher Scientific) followed by staining with Imperial protein stain (Thermo Fisher Scientific) according to manufacturer's instructions.

Ethics statement

Historic patient specimens received at the CDC Rabies Laboratory for antemortem rabies diagnostic testing were used in the validation of the N protein ELISA. The samples originated from facilities in the United States (U.S.) where the patients were being hospitalized and treated. Samples were de-identified according to IRB approved CDC protocol #7028. A formal request to waive consent was approved by the CDC IRB.

Clinical specimens

CSF and serum specimens collected from patients (January, 2005 to January, 2018) suspected of rabies were originally tested by the IFA test and RFFIT for binding and neutralization antibody detection, respectively. A total of 65 serum specimens and 46 CSF specimens from 16 rabies-diagnosed cases were tested in our study along with 50 sera and 30 CSF from rabies negative human rule-out patients, including patients exhibiting acute progressive encephalitic symptoms of unknown etiology. However, in some instances sample volume was limited or previous sample information was missing and only a subset of samples were included in a particular analysis. Samples from rabies and non-rabies cases were heat inactivated at 56°C for 45 minutes and evaluated for anti-N antibody detection by ELISA. Healthy sera from four unvaccinated individuals and one serum sample from a non-rabies case with a confirmed alternative diagnosis were used as controls. These samples were confirmed negative by both the IFA test and RFFIT prior to use. CSF negative controls were from non-rabies cases, in which there was an alternative diagnosis and samples were confirmed negative by the IFA test prior to use. Positive samples that were used as internal assay controls included standard rabies immune globulin (SRIG, Lot R-3) diluted to 2 IU/ml and an IFA positive human serum sample from a previous rabies positive case.

Indirect fluorescent antibody (IFA) test

The IFA test was performed on clinical samples as part of rabies antemortem diagnostic testing at the CDC according to Clinical Laboratory Improvement Amendments of 1988 (CLIA) standards. For confirmation, repeat testing was done on a selected number of samples using the IFA protocol as described previously [ 37]. In brief, mouse neuroblastoma (MNA) cells (0.5x10 ⁶ cells/ml) were infected with RABV CVS-11 to obtain a target of 25 to 50% infected cells. The virus-cell suspension was plated drop-wise onto 4-well 6 mm Teflon ®-coated microscope slides (Thermo Fisher Scientific) using a disposable dropper, yielding 1 drop per well. Slides were incubated in a moist chamber at 37°C with 0.5% CO ₂ for 20 hours to allow for infection and cell adherence. After incubation, wells were rinsed with 1x 0.01 M PBS and fixed with cold acetone for 30 minutes. Infection was confirmed by immunofluorescence using a monoclonal anti-N protein fluorescein isothiocyanate (FITC)-conjugated antibody (Fuijirebio Diagnostics, Inc) and visualized using an Axio Imager A1 fluorescence microscope (Zeiss) at 200X magnification. Heat-inactivated diluted serum or undiluted and diluted CSF were added to the wells and incubated for 30 minutes at 37°C, then rinsed twice with 1x PBS for 5 minutes. The secondary FITC-conjugated antibodies, goat anti-human IgG (H+L) (SeraCare) or anti-human IgM (SeraCare), were added and incubated at 37°C for 30 minutes, then rinsed twice for 5 minutes with 1x PBS. The slides were mounted with 20% glycerol and visualized with a fluorescence microscope. If no specific RABV inclusions were detected in the sample and the negative control, the sample was reported to have a negative IFA result. If the sample had FITC fluorescent staining of typical inclusions within cells as demonstrated in the positive control, the test was reported as positive.

Confocal microscopy and imaging

Slides were prepared similarly to the IFA test procedure, in which a suspension of MNA infected cells were seeded on Teflon ®-coated slides to make a monolayer, fixed with acetone and used for further staining. Fixed infected cells were blocked with 10% fetal bovine serum (FBS) in 1x PBS for 15 minutes at room temperature (RT). Mouse anti-N mAbs and human sera were diluted in 1x PBS with 10% FBS and incubated alone or in combination for 30 minutes at RT. Wells were then washed with 1x PBS multiple times. Alexa Flour 594 conjugated anti-mouse (Molecular Probes) and FITC-conjugated anti-human IgG (SeraCare) in 1x PBS were used as secondary antibodies. Secondary antibodies were incubated for 30 minutes at RT. Cell nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI). The cells were then fixed with 4% paraformaldehyde for 10 minutes, followed by mounting with Prolong Antifade Mounting Media (Thermo Fisher Scientific). The cells were then visualized using a LSM 710 inverted confocal microscope (Zeiss).

N protein IgG & IgM ELISA

Immunlon II high binding microtiter 96-well plates (Thermo Fisher Scientific) were coated with purified N protein diluted in carbonate-bicarbonate buffer (Sigma-Aldrich) overnight at 4°C. Protein coating concentration for IgG detection was 0.125 μg/ml and 0.50 μg/ml for IgM. The plates were washed three times with 0.05% PBS/Tween (PBS-T) between each incubation step and all incubations were at RT. Plates were blocked for 1 hour with blocking buffer (5% skim milk, 2% bovine serum albumin, and 2% goat serum in 0.05% PBS-T) at RT. CSF samples were diluted at 1:10 and serum samples were diluted at 1:100 in blocking buffer and incubated on the coated plate for 1 hour. Secondary goat anti-human IgG-horseradish peroxidase (HRP) conjugated antibody (SeraCare) was diluted in blocking buffer at 1:2000 and goat anti-human IgM-HRP conjugated antibody was diluted at 1:6000 (SeraCare), then added to the plates for 1 hour. To develop the samples, 3,3',5,5' Tetramethylbenzidine (TMB) 2-component substrate (Sera Care) was added to the plates, followed by TMB Stop Solution (SeraCare). The ODs were measured at 450 nm using the Enspire Plate Reader (PerkinElmer). In addition, two mouse anti-RABV N conformation-specific mAbs, clone CDC 62-15-2 and clone CDC 62-164-2 [ 38] were used as the primary antibody to detect specific epitopes using our ELISA format and anti-mouse IgG-HRP was used as the secondary detection antibody.

Data analysis

Every plate contained two positive and three to five negative control samples. The negative control samples were used to determine the cutoff value (COV) for individual plates. All samples were tested in duplicate. The average OD value of the blank wells (blocking buffer only) was subtracted from control and test samples before the COV was calculated. The COV corresponded to the mean OD of all negative control samples plus three times the standard deviation. The COV was then subtracted from the average of each test sample's OD value to determine positive and negative results, which were the reported OD values. Statistical analysis was performed on the calculated OD values from IFA positive and negative samples in GraphPad Prism software using the Mann-Whitney test. Statistics were reported as P values. Sensitivity and specificity percentages were calculated for each N protein ELISA test using the IFA results as the true positive and negative values. Sensitivity was calculated by the following formula (true positive/(true positive + false negative)). Specificity was calculated using the following formula (true negative/(false positive + true negative)). The 95% confidence intervals for sensitivity and specificity were also reported and are Clopper-Pearson confidence intervals.

Results

Rabies positive human serum detects N in RABV infected cells

To demonstrate that the rabies positive human serum primarily detects N protein in an IFA assay, we first compared the IFA staining pattern of rabies positive human serum samples with that of anti-N protein staining in RABV-infected cells. RABV-infected cells were prepared on slides similarly to an IFA assay and incubated with either human rabies IFA positive serum or non-rabies IFA negative serum and mouse anti-N protein mAb. N protein and human serum staining were visualized by confocal imaging using anti-mouse IgG Alexa Flour 594 or anti-human IgG FITC conjugated antibodies. As expected, we observed specific staining with serum from a positive human rabies case and no staining with negative human serum ( Fig 1, compare top and bottom left panels). The staining pattern observed with the positive serum localized with the anti-N protein mAb staining ( Fig 1, merge), confirming the predominant detection of N protein by human serum in an IFA positive sample. This result suggested the potential use of an N protein-based ELISA for the detection of anti-RABV binding antibodies in human cases.

Recombinant RABV N expression, purification and conformational analysis

Recombinant N protein from RABV CVS-11 was produced using an E. coli expression system. The overall purity of the N protein sample was evaluated by SDS-PAGE followed by staining with Imperial protein stain, a Coomassie blue staining for protein visualization ( Fig 2A). The staining revealed a single prominent band at approximately 50kDa, suggesting that the N protein expressed was successfully isolated and was relatively free of other proteins.

To determine if the recombinant N protein retained native conformational epitopes, we used two mouse mAbs against N protein that were previously characterized to bind distinct conformation-specific epitopes [ 38- 40]. Both mAbs demonstrated a concentration-dependent binding to recombinant N protein compared to the mouse IgG isotype control ( Fig 2B). Since these mAbs are used routinely as primary antibodies for rabies antigenic typing in the IFA test, we are confident that the expressed N protein closely resembles native conformation. Hence, the recombinant N protein is a suitable antigen to detect binding of polyclonal antibodies generated in humans infected with RABV.

N protein IgG and IgM ELISA optimization for serum and CSF

Using the purified recombinant N protein, we developed an indirect ELISA for the detection of IgG and IgM antibodies against RABV N protein in human CSF and serum specimens. The coating antigen concentration, sample dilution, and secondary antibody concentration were optimized using either SRIG, human sera, or CSF from rabies and non-rabies cases. SRIG, in addition to specimens from actual human cases, was used for optimization since it is a standardized preparation of rabies immune globulin with known concentration. Human specimens (serum or CSF) from rabies and non-rabies cases allowed for comparison of ELISA values to determine the background signal. We first determined the coating concentration and serum dilution for the IgG ELISA by narrowing our coating antigen range to 0.125-1.000 μg/ml and serum dilution from 1:100 to 1:400 for both positive and negative samples ( S1A-S1C Fig). The ratio of positive (SRIG or rabies signal) to negative signal (non-rabies) was also calculated ( S1D and S1E Fig). Since a decrease in positive to negative signal ratio was observed at increasing coating concentrations, 0.125 μg/ml of N protein and a serum dilution of 1:100 was used for subsequent experiments. Using a serum dilution of 1:100 optimized by the IgG ELISA, the N protein concentrations and anti-IgM secondary dilutions for the IgM ELISA were varied ( S2 Fig). Since SRIG is not positive for the IFA IgM test, it served as a negative control in optimization, further substantiating the notion that detection of IgG antibodies are specific for N protein in the ELISA. The N protein coating concentration and secondary dilution that yielded optimal optical density (OD) measurements for the IgM ELISA was determined to be 0.5 μg/ml and 1:6000, respectively ( S2 Fig).

To maintain consistency in our IgM and IgG ELISAs, concentrations as described above for coating and secondary antibody were used for CSF samples. The CSF sample dilution for both IgG and IgM was determined by titrating 3 rabies CSF positive samples and 2 non-rabies negative samples from 1:10 to 1:100 ( S3 Fig). The background for CSF negative samples was relatively low at all dilutions. Therefore, to detect the largest difference in OD measurements between positive and negative samples and also capture any low positive samples, a 1:10 dilution of CSF samples were used for both IgG and IgM.

Comparison of RABV N protein ELISA with the IFA test

Individual serum and CSF specimens from previous rabies and non-rabies diagnosed cases from 2005 to 2018 were tested for RABV N protein-specific antibodies using the N protein ELISA. A total of 77 sera and 39 CSF samples were tested for N protein IgG and 79 sera and 38 CSF samples were tested for N protein IgM, including the negative samples from non-rabies cases ( S4 Fig). To check the stability of anti-RABV binding antibodies upon storage, IFA testing was repeated in a subset of samples and we observed consistent results to those previously reported. The cut off values (COV) were determined for each plate based on the OD values and standard deviation of the negative control samples. The OD values for both IgM and IgG rabies positive CSF samples varied widely in the ELISA ( Fig 3A and 3B). Some samples had values just above the COV and others as high as 2.0, demonstrating differences in immune responses between the rabies cases. However, the average OD values for rabies CSF positive samples versus non-rabies negative samples was statistically significant for both IgM and IgG. Among the rabies IFA negative CSF samples, only three (1 IgM and 2 IgG) samples were higher than the COV value. The ELISA results for rabies positive serum samples also varied in terms of OD values ( Fig 4A and 4B). Even though the average IgM and IgG OD values between positive and negative were statistically significant ( Fig 4A and 4B), a number of false negative and false positive ELISA results were observed ( Fig 4C and 4D).

The sensitivity and specificity of N Protein ELISA were calculated based on the IFA result as the true positive and negative. Although the readout of two assays is different (absorbance versus microscopy), there was a high correlation observed for both IgM and IgG in the CSF ( Fig 3C and 3D). The sensitivity and specificity of CSF IgM ELISA for rabies and non-rabies cases were 100% and 96.77%, respectively when compared to the IFA result. Similarly, the CSF IgG ELISA had 90% sensitivity and 93.75% specificity. The sensitivity and specificity values for serum IgM and IgG results relative to the IFA results, however, were lower compared to the tests using CSF. The serum IgM ELISA exhibited 78.38% sensitivity and 94.34% specificity ( Fig 4C), while serum IgG ELISA demonstrated 81.82% sensitivity and 75.76% specificity ( Fig 4D). We presume one of the reasons for the higher proportion of discordant results in serum compared to CSF may be due to higher non-specific binding. Even in the IFA test, serum exhibits a higher background compared to corresponding CSF samples from a single patient.

N protein ELISA results for rabies classification

We further evaluated the N protein ELISA by comparing the results to the IFA results of independent rabies cases. As previously mentioned, a rabies diagnosis is determined by testing a variety of samples with different methods. Hence, the outcome of the ELISA results does not change the overall interpretation of a previous rabies diagnosis. The IFA and ELISA results were compared in a total of 16 individual rabies cases with exposure to a rabid bat or dog via bite, contact, or unknown contact. The clinical presentation of each patient varied, with symptoms including encephalitis, fever, headache, agitation and combativeness, paresthesia, and hydrophobia ( S1 Table). In this analysis, all samples from a single patient were treated as a set and we determined if the patient had at least one positive specimen for RABV antibodies by the IFA test or N protein-specific antibodies by ELISA during the course of the disease ( Fig 5A). The number of specimens from a single rabies diagnosed individual ranged from 1 to 14 for either serum or CSF ( S1 Table).

Seven rabies-diagnosed cases were reported to have an IgM response by IFA in CSF specimens and of those 7 cases, 6 cases had an N protein ELISA positive result, and 1 case we could not obtain data due to limited availability of sample volume ( Fig 5B). Of the 9 cases that had a previous negative IgM IFA result in the CSF, N protein ELISA was positive for 4 cases, 1 case was negative and we could not obtain data for the remaining 4 cases. In terms of the IgG IFA results for CSF specimens, 8 cases were previously positive and 8 were previously negative. Of the 8 IFA positive cases, 5 were positive by IgG N ELISA, 2 were negative and for 1 sample we could not obtain data. For the IFA negative cases, N protein ELISA detected one positive, 3 negative, and 4 cases did not have sufficient sample to test. Overall, 10 of 11 cases (based on sample availability) were positive for IgM and 6 of 11 were positive for IgG using N protein-specific ELISA in the CSF.

For the serum, 13 rabies-diagnosed cases were positive for IgM by the IFA test and 3 cases were negative. For IgG, 12 cases were previously positive and 4 cases were previously negative for IgG by the IFA ( Fig 5A). Of the 13 IFA IgM positive cases, 10 were positive by IgM N protein ELISA and 3 were negative ( Fig 5B). From the 3 negative IFA IgM cases, all cases were also negative in the N protein ELISA. For serum IgG, of the 12 IFA positive cases, all 12 were also positive in the N protein ELISA and of the 4 IFA negative cases, 3 were negative and 1 was positive by N protein ELISA. Overall, 10 of 16 cases had N protein IgM seroconversion and 13 of 16 cases had N protein IgG seroconversion. The N protein ELISA results for serum from rabies-diagnosed cases was mostly consistent with previous IFA results and the N protein ELISA was able to detect an additional IgG positive serum sample ( Fig 5).

Kinetics of anti-N antibody detected in rabies-diagnosed cases

We examined the kinetics of anti-N protein IgG and IgM antibodies in the CSF and serum of several individual rabies cases. A total of 5 cases for CSF and 6 cases for serum were evaluated based on days from illness onset to the sample collection date, which varied from case to case. Antibodies in the CSF were detected after day 10 of illness onset ( Fig 6) and seen as late as Day 37. In all cases, only one of the two antibody subclasses was detected in CSF. For instance, in rabies cases 3, 4, 14 and 16, only IgM antibodies were detected ( Fig 6A, 6B, 6C and 6E), whereas in case 15 only IgG antibodies were detected ( Fig 6D). In contrast, the serum IgG and IgM profiles for 3 rabies cases showed seroconversion of IgM before IgG ( Fig 7A, 7C and 7D) and 3 cases only IgM or IgG was detected ( Fig 7B, 7E and 7F). The earliest serum antibodies were detected was on Day 8 ( Fig 7C) and the latest was on Day 32 ( Fig 7B). Interestingly, for rabies case 15, only IgG was detected in both CSF and serum, but no IgM. In cases 3 and 8, the onset and progression of IgM and IgG antibody profiles exhibited a similar pattern.

Discussion

Antemortem diagnosis of rabies is challenging due to the clinical course of the disease that can be influenced by viral pathogenesis and immunological status [ 41]. Laboratory assays rely on the dissemination of the virus for the detection of nucleic acid, antigen, and RABV-specific antibodies, which can depend on intermittent viral excretion [ 42]. The World Health Organization (WHO) recommends the collection of several specimen types over the course of illness in order to perform a variety of standard diagnostic tests including those for virus isolation, antigen, RNA and antibody detection [ 42]. However, this becomes increasingly difficult in countries with limited resources to collect the appropriate samples and have the equipment to perform the recommended assays. Therefore, development and evaluation of accessible assays are important for determining the true burden of disease. Here, we evaluated an ELISA-based assay for detection of RABV N protein-specific antibodies in human serum and CSF specimens from previously diagnosed rabies cases.

Antemortem diagnosis of rabies at the CDC requires 4 specimen types (saliva, serum, CSF, and skin) [ 43] to perform antigen detection using DFA [ 31, 32], nucleic acid detection using RT-PCR [ 44- 46], and antibody detection using IFA [ 37] and RFFIT [ 13]. RABV antibody detection is informative for determining immune status and previous exposure to RABV antigens. This information can be useful not only in the diagnosis of rabies but also in surveillance, vaccine immunogenicity or research studies. At CDC, the two methods used for antibody detection in human antemortem testing are the IFA test and the RFFIT. In addition, the fluorescent antibody virus neutralization (FAVN) test [ 43, 47, 48] and the ELISA are also standard human antemortem antibody detection tests suitable for serum and CSF [ 42, 47]. ELISA-based assays for determining antibody response after vaccination utilize predominantly G protein [ 18- 22] as the target antigen since it is an exposed viral surface protein and highly immunogenic. However, viral N protein is also highly immunogenic and reported to enhance immunity and virus neutralizing antibody activity [ 3, 4, 10]. A few studies have investigated N protein-specific antibody responses in humans using N protein as the target antigen for ELISA-based assays [ 23- 26]. Here, we developed an N protein ELISA and validated it using samples that were previously confirmed as positive or negative for the presence of anti-RABV binding antibodies by the standard IFA test. This study demonstrated RABV N protein-specific responses in human CSF and anti-N seroconversion with both IgG and IgM subtypes in a single patient over the course of illness using an N protein ELISA.

One of the advantages of performing a binding assay compared to a neutralization test is the ability to identify Ig subclass types, such as IgG and IgM. While IgG will generally indicate a previous exposure to RABV antigen, the IgM develops before IgG during the acute phase of a recent infection. Anti-RABV antibodies on average are detected in the serum or CSF of rabies patients on day 7 or 10.5, respectively, from illness onset and as early as 2 days [ 49]. The primary objective of this study was to investigate the use of recombinant N protein for binding antibody detection in clinical samples diagnosed with RABV infection. We developed an indirect ELISA to detect either IgM or IgG antibodies generated against RABV N protein following an infection in CSF and serum samples, which are two of the four sample types used in the antemortem diagnosis of rabies. We demonstrated detection of both IgM and IgG anti-N antibodies in CSF and serum in several previously diagnosed human cases and also show the kinetics of antibody detection in individual rabies cases in which multiple samples were available. From the cases that had detectable N protein-specific antibodies, there was variability in the time of illness onset to the time of antibody detection and also in the profiles of antibody response. For instance, in one case the IgM response in the CSF was detected as early as day 13 but reached a peak at day 16 and dropped by day 17. In another case, there was an IgG but no IgM response detected in the CSF, although the IFA test did not detect any CSF antibody responses. The kinetics of antibody responses in the serum was generally observed as expected. Detection of IgM was followed by IgG, but in two instances IgG was detected with no IgM. IgG detection in the absence of IgM has previously been reported for rabies [ 50]. However, it is possible that IgM was below the detection in both the ELISA and the IFA in these particular samples. In some instances, no antibodies were detected throughout the course of illness, especially in the CSF [ 49, 51, 52].

The kinetics of antibody production, in addition to antigen (N protein) and genomic RNA detection, varies in rabies cases. Therefore, performing a variety of tests can increase the likelihood of obtaining a positive antemortem diagnosis [ 49, 53]. Antigen detection (DFA) and genomic RNA (RT-PCR) -based assays are also important for rabies antemortem diagnostics and are performed as part of the standard diagnostic tests [ 42] in conjunction with antibody detection assays. Although antigen and genomic RNA can be detected initially in nuchal skin biopsy and saliva samples, antibodies in the serum or CSF may not be detected for several days after the onset of symptoms [ 41, 49, 50, 54]. In animal studies, generation of antibodies can enhance infection and results in 'early death' due to complement-mediated opsonization of immune complexes [ 55, 56], however, the role of immunopathology in positive human cases is not clearly defined. Conversely, in some cases of survival, patients had higher levels of neutralizing antibodies in the serum and CSF early on and antigen or genomic RNA was not detected in nuchal skin biopsy and or in saliva samples [ 50, 57- 59]. This further emphasizes the importance of collecting multiple antemortem samples (nuchal skin biopsy, serum, CSF and saliva) for making a proper diagnosis and potential prognosis.

In this study, we compared the performance of N protein ELISA with IFA for detection of binding antibodies. In the N protein ELISA, CSF samples exhibited high sensitivity and specificity in both IgM and IgG assays (greater than 90%) when considering the IFA results as the true positive and true negative. The IgG and IgM ELISA tests using serum samples exhibited a lower sensitivity and specificity compared to the tests using CSF, but the N protein ELISA was still able to detect both IgM and IgG subclasses in a majority of rabies samples that were previously IFA positive. The discordance between the ELISA and IFA assays may be due to differences in assay conditions and the method of detection. While the IFA test can detect RABV-specific antibodies against all viral proteins encoded by RABV, the ELISA detects only N protein-specific antibodies. Therefore, a positive IFA and a negative ELISA result for a particular sample may suggest the presence of antibodies against other proteins, particularly G protein. In addition, the IFA test has the advantage of being able to visually examine the pattern for anti-RABV- specific versus non-specific staining, which could identify false positives. However, even with the IFA test, false positive results have been observed due to cross-reactive antibodies from encephalitic patients infected with flaviviruses [ 29], which is a limitation to consider when performing antibody binding assays. Similarly, the false positive rate, particularly in the serum IgG ELISA, can lower the positive predictive value. Therefore, exposure history, clinical symptoms, and results from additional confirmatory testing (if available) should be taken into consideration in conjunction with the N protein ELISA. One of the limitations of our study is the availability of samples from human rabies positive cases in the U.S. [ 60]. From a total of 41 cases were reported in the U.S. and Puerto Rico from 2003 to 2018 [ 49, 60], only 16 had both CSF and serum samples available with sufficient volume to test for N protein-specific antibodies. Collaboration with laboratories in rabies endemic countries would help to improve optimization, accuracy, positive predictability and repeatability of the assay.

In addition to individual samples, we also evaluated the overall ability of the ELISA to detect N protein-specific antibodies in any sample from 16 previously diagnosed rabies cases compared to the IFA. Although the results for both IFA and ELISA are comparable, there were some instances in which one test detected an antibody response and the other did not. For example, in cases 3, 15, and 16, although IFA did not detect either IgM or IgG in CSF, ELISA was positive for IgM or IgG ( Fig 5A). While cases 7 and 14 did not seroconvert for IgM nor IgG in CSF or serum by IFA, the N protein ELISA was able to detect IgG in the serum (case 7) and IgM in the CSF (case 14) of those respective patients. On the contrary, there were instances in which IFA was positive and ELISA was negative, particularly with serum IgM results (cases 4, 13 and 15) and CSF IgG results (case 4, 8). Overall, the N protein ELISA was able to detect antibodies in 15 out of the 16 rabies cases (94%), whereas the IFA detected antibodies in 13 out of 16 rabies cases. The results suggest that N protein could be sufficient for the detection of RABV binding antibodies, even though other RABV protein-specific antibodies, such as G protein-specific antibodies, could also be present.

An ELISA based detection of anti-N antibodies offers several advantages compared to the IFA test. Since the recombinant N protein used in the ELISA is purified from E. coli and antibodies are detected by a colorimetric method, it could be adopted in a hospital or resource-limited setting. In contrast, IFA requires RABV-infected cells that are not fully inactivated by standard fixation methods [ 37]. In addition, the ELISA does not require a fluorescence microscope, fluorescence detection reagents, or a highly trained individual to differentiate RABV-specific and non-specific fluorescence staining. In that respect, the ELISA gives an opportunity to standardize an assay globally across different labs, allowing for direct comparability of anti-N IgM and IgG kinetics. However, further validation is required and implementation of the N protein ELISA is recommended in conjunction with other standard antemortem diagnostic tests. The ELISA also offers the opportunity to test samples in a high-throughput format compared to the IFA test. Therefore, it can be optimized for non-diagnostic purposes, such as animal surveillance studies or serosurveys.

Acknowledgments

The authors thank the physicians, laboratorians, epidemiologists, and affiliates with public health departments and hospitals that assisted in the diagnosis of reported rabies cases and sample submissions to the CDC. We thank Mary Reynolds, Kim Hummel, Michael Townsend, James Peterson and the reviewers for valuable comments and suggestions. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the CDC.

References

59. Jean Wiedeman JP, Glaser Carol, Messenger Sharon, Wadford Debra, Sheriff Heather, Fritz Curtis, Lindsay Ann, McKenzie Mary, Hammond Christina, Gordon Eric, Rupprecht Charles E. Recovery of a patient from clinical rabies-California, 2011. MMWR Morb Mortal Wkly Rep. 2012;61(4).

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An ELISA-based method for detection of rabies virus nucleoprotein-specific antibodies in human antemortem samples

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Rabies is a fatal encephalitic disease in humans and animals caused by lyssaviruses, most commonly rabies virus (RABV). Human antemortem diagnosis of rabies is a complex process involving multiple sample types and tests for the detection of antibodies, antigen (protein), and nucleic acids (genomic RNA).

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 ELISA Test For The Serological Detection Of Scedosporium/Lomentospora In Cystic Fibrosis Patients

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 ELISA Test For The Serological Detection Of Scedosporium/Lomentospora In Cystic Fibrosis Patients

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Martin-Souto, Leire, Idoia Buldain, Maialen Areitio, Leire Aparicio-Fernandez, Aitziber Antoran, Jean-Philippe Bouchara, Maria Teresa Martin-Gomez, Aitor Rementeria, Fernando L. Hernando, and Andoni Ramirez-Garcia. 2020. "ELISA Test For The Serological Detection Of Scedosporium/Lomentospora In Cystic Fibrosis Patients". Frontiers In Cellular And Infection Microbiology 10. doi:10.3389/fcimb.2020.602089.

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Abstract

The detection and diagnosis of the opportunistic fungi Scedosporium spp. and Lomentospora prolificans still relies mainly on low-sensitive culture-based methods. This fact is especially worrying in Cystic Fibrosis (CF) patients in whom these fungal species are frequently isolated and may increase the risk of suffering from an infection or other health problems. Therefore, with the purpose of developing a serologic detection method for Scedosporium/ Lomentospora, four different Scedosporium boydii protein extracts (whole cell protein extract, secretome, total cell surface and conidial surface associated proteins) were studied by ELISA to select the most useful for IgG detection in sera from CF patients. The four extracts were able to discriminate the Scedosporium/ Lomentospora-infected from Aspergillus-infected and non-infected patients. However, the whole cell protein extract was the one selected, as it was the one with the highest output in terms of protein concentration per ml of fungal culture used, and its discriminatory capacity was the best. The ELISA test developed was then assayed with 212 sera from CF patients and it showed to be able to detect Scedosporium spp. and Lomentospora prolificans with very high sensitivity and specificity, 86%-100% and 93%-99%, respectively, depending on the cut-off value chosen (four values were proposed A 450nm= 0.5837, A 450nm= 0.6042, A 450nm= 0.6404, and A 450nm= 0.7099). Thus, although more research is needed to reach a standardized method, this ELISA platform offers a rapid, low-cost and easy solution to detect these elusive fungi through minimally invasive sampling, allowing the monitoring of the humoral response to fungal presence.

enzyme linked immunosorbent assay, Keywords: Scedosporium, cystic fibrosis, serodiagnosis, Lomentospora

Highlights

In recent years huge efforts have been made to develop new serological techniques to improve diagnosis of fungal infections. However, most of the advances are focused on high prevalence fungal pathogens such as Aspergillus or Candida. Regarding less common fungi like Scedosporium/Lomentospora, which are considered emerging pathogens and are gaining clinical relevance due to the severity of the infections they cause, there are no commercial detection methods available. This manuscript describes an ELISA test developed using a whole cell protein extract from Scedosporium boydii that offers a great sensitivity and specificity to detect Scedosporium/Lomentospora in Cystic Fibrosis (CF) patients using serum samples. Compared with the commercial kits for other fungi, which usually show a 75%-96% of sensitivity, the clinical usefulness of the test developed is remarkable considering that values obtained are of 86%-100% for sensitivity and 93%-99% for specificity. These results place it in a good position as a candidate to be used as a diagnostic tool. Thus, this ELISA platform is a rapid, low-cost, and easy solution to detect Scedosporium/Lomentospora, allowing the monitoring of the antifungal humoral response.

Introduction

Cystic fibrosis (CF) is the major genetic disorder among the Caucasian population ( Mirtajani et al., 2017). This multisystem disease is caused by mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene encoding a chloride-conducting transmembrane channel, which participates in electrolytic transport and mucociliary clearance of the airways. CFTR dysfunction results in an increased viscosity of secretions and underlies an altered immune response of these patients ( Rowe et al., 2005). Although several organs are adversely affected, morbimortality is essentially associated with lesions in the lungs ( Castellani & Assael, 2017). The airways of CF patients are affected by the accumulation of a thick layer of sticky bronchial mucus, which acts as a culture media for microorganisms, entrapping airborne bacteria and fungal spores, immobilizing them and facilitating their growth. In this sense, these pathogens cause chronic respiratory infections, turning the lung into an inflammatory microenvironment and eventually leading to pulmonary damage ( Elborn, 2016).

Even though bacteria, such as Pseudomonas aeruginosa or Staphylococcus aureus, are known to be the major causative agents of these infections, several fungal species colonize the respiratory tract of CF patients. However, while the relevance of bacteria is well known, the clinical significance of fungal recovery from respiratory secretions remains unclear ( Schwarz et al., 2018). Candida albicans, among yeasts, and Aspergillus fumigatus, among filamentous fungi, are the fungal species most frequently isolated from CF respiratory samples. However, fungi from Scedosporium genus are increasingly reported in the CF context and currently rank second, just behind A. fumigatus, among the filamentous fungi colonizing CF airways ( LiPuma, 2010).

Despite airway colonization by Scedosporium spp., or the strongly related Lomentospora prolificans ( Ramirez-Garcia et al., 2018), it is usually well tolerated, it may lead to a true respiratory infection with variable degree of tissue invasion, fungal sensitization or allergic bronchopulmonary mycoses ( Martín-Gómez, 2020). In fact, these fungal pathogens seems to be more representative during scenarios of moderate-to-severe alteration of lung function, and their presence on CF airways has been associated with a decline in Forced Expiratory Volume in 1 s (FEV1) ( Soret et al., 2020). In addition, the chronic detrimental presence of these pathogens may cause fatal disseminated infections when the patient undergoes an immunosuppression period, for example after a lung transplantation ( Symoens et al., 2006).

Unfortunately, lung infection or colonization by Scedosporium/ Lomentospora is nowadays a diagnostic and therapeutic challenge in CF patients. Despite the many methods of Aspergillus detection being currently available, the detection of Scedosporium/Lomentospora relies upon low sensitivity culture-based traditional methods. Recently, some advances have been made on molecular diagnosis, but these methodologies are not standardized and not accessible to everyone, and serodiagnosis strategies are performed only in specialized laboratories, but these are not commercially available ( Mina et al., 2017). Therefore, the precise diagnosis of these fungi is actually a significant challenge. Furthermore, the clinical features and histopathology of infected tissue samples are similar to those of aspergillosis, so confirmation data of these species may often be underestimated ( Mello et al., 2019). Hence, detection and correct discrimination of Scedosporium infections from others is of crucial importance because the treatments may be quite different. Indeed, Scedosporium/ Lomentospora species are considered intrinsically resistant to most of the currently available antifungal drugs ( Pellon et al., 2018).

In this sense, to contribute to the finding of new diagnostic weapons that allow prevention, early diagnosis, and ultimately a more effective treatment, our research group analysed different protein extracts of Scedosporium boydii as serodiagnostic tools to detect Scedosporium/Lomentospora and discriminate them from other fungal pathogens relevant in the CF context. Therefore, in this study we designed and tested a customized serological assay for the detection of Scedosporium-specific IgG antibodies in sera from CF patients.

Materials and Methods

Microorganisms and Culture Conditions

The fungal strains used in this study were Scedosporium boydii CBS 116995, Lomentospora prolificans CECT 20842, Aspergillus fumigatus Af293 and Candida albicans NCPF 3153. All strains were maintained cryopreserved at -80ºC and cultured as required on Potato Dextrose Agar (PDA) (Pronadisa, Madrid, Spain).

To harvest conidiospores of S. boydii and L. prolificans, PDA plates grown at 37ºC for 7 days were washed twice with sterile saline solution (0.9% [w/v] NaCl) (SS). The suspension of conidia was filtered through sterile gauze to avoid cell debris and centrifuged. Conidia of A. fumigatus were collected from PDA tubes grown at 37ºC for 4 days using sterile SS-Tween20 (0.9% [w/v] NaCl, 0.02% [v/v] Tween20), and washed twice by centrifugation. Finally, C. albicans was grown in PDA tubes at 37ºC for 24 h, and yeast cells were gathered next day by resuspending the culture with phosphate buffered saline (PBS). The concentration of each fungal cell was adjusted as needed using a hemocytometer.

Human Serum Sample Collection and Categorization

A collection of 212 sera from CF patients (corresponding to 102 different patients) were used in this study with the approval of the Ethics Committee from the University of the Basque Country (UPV/EHU; reference M30/2018/081).

The categorization of the sera was based on the results of mycological examination of a sputum sample collected in parallel to the sera and inoculated on Sabouraud gentamicin chloramphenicol agar, and simultaneously on Modified Thayer Martin agar or Sabouraud chloramphenicol agar supplemented with 0.5 g/L cycloheximide for the specific recovery of Scedosporium species from these polymicrobial samples. All plates were incubated at 37ºC for up to 15 days before to consider the sputum samples as free of fungi. According to these, three groups of sera were defined: Group Scedo+ (n = 23) consisted of sera from CF patients with positive cultures for Scedosporium/ Lomentospora, Group Asp+ (n = 86), CF patients with Aspergillus spp. being the only filamentous fungi recovered from sputum; and group Scedo-/Asp- (n = 103) as control, consisted of sera from CF patients without any filamentous fungus recovered from samples. Sera from patients with co-infection of Scedosporium/ Lomentospora and Aspergillus spp. were included in the group Scedo+.

In addition, five sera from each group described above were selected to evaluate the usefulness of different protein extracts for Scedosporium/ Lomentospora serodiagnosis, and to study cross-reactivity with other fungal pathogens. To do that, two criteria were followed: each serum corresponded to a different patient without any coinfections.

Fungal Protein Extracts

Four different protein extracts were obtained for this study: whole cell protein extract (Total WCP), extract of secreted proteins (Secretome), cell surface associated proteins (Total CSP) and conidial surface proteins (Conidial CSP). All extracts were obtained for S. boydii, but the Total WCP was also obtained for L. prolificans, A. fumigatus, and C. albicans. The resulting protein extracts were stored at -80ºC until required.

The extraction processes () were carried out in triplicate and the quality of the extract was verified by SDS-PAGE in 12% polyacrylamide gels, stained afterwards as previously described ( Dyballa and Metzger, 2009) with Coomassie Brilliant Blue G250 (CBB), and digitalized using ImageScanner III (GE Healthcare, Chicago, IL, USA). Protein concentration was quantified using Pierce 660 nm Protein Assay Reagent (Thermo Fisher Scientific, Rockford, IL, USA). Likewise, to avoid interference from some reagents present in the extraction buffers, protein extracts were precipitated with a solution of acetone and 10% (w/v) trichloroacetic acid, and resuspended in distilled water, prior to protein concentration measurement.

Whole Cell Protein Extract of Conidia and Hyphae (Total WCP)

To obtain total protein extracts, 5 x 10 ⁵ cells/ml (conidia or yeasts) were inoculated into Potato Dextrose Broth (PDB) (Pronadisa, Madrid, Spain) cultured at 37ºC and 120 rpm for 24 h. Fungal growth was recovered by filtration and washed twice with PBS to remove traces from the medium. Fungal material was resuspended in PBS supplemented with 1% (v/v) β-mercaptoethanol and 1% (v/v) ampholytes pH 3-10 (GE Healthcare, Freiburg, Germany). Finally, cell disruption was achieved by bead-beating with glass beads for 20 min at 30 Hz using the MillMix20 (Tethnica, Slovenia), following the standardized protocol described previously ( Pellon et al., 2016). Cell debris was discarded by centrifugation, and the resulting protein suspension was sonicated on ice for 2 min at 40% amplitude and 2 s pulses.

Extract of Secreted Proteins (Secretome)

Extraction of S. boydii secretome was carried out following the methodology described in Buldain et al., 2019, with slight modifications. Briefly, 10 ⁶ conidia/ml were inoculated into PDB and grown for 24 h at 37ºC and 120 rpm. Fungal material was collected, washed twice with sterile PBS and cultured for 20 h at 37ºC and 120 rpm in PBS supplemented with 2% glucose in a proportion 1 g fungus: 1 mL medium. The culture was centrifuged, the supernatant filtered through a sterile gauze and then through a 0.22 µm membrane. Finally, the cell-free supernatant was concentrated using a 100,000 MWCO VIVASPIN centrifugal concentrator system (Sartorius, Göttingen, Germany). The resulting suspension was sonicated under the same conditions as the Total WCP extract described above.

Cell Surface Associated Proteins (Total CSP and Conidial CSP)

Proteins associated with the cell surface were collected by employing the protocol described by Pitarch et al. (2002), but with some modifications. To be precise, the culture of 5 x 10 ⁵ conidia/ml PDB for 24 h at 37ºC and 120 rpm was recovered by filtration and washed twice with PBS. Fungal material was resuspended in a volume of SDS extraction buffer and boiled for 10 min at 100ºC, cooled 5 min on ice, centrifuged and the supernatant containing Total CSP was recovered.

To obtain the Conidial CSP extract, the same procedure was carried out but using 10 ⁸ conidiospores as fungal material, which were directly resuspended in 1 ml of extraction buffer.

Enzyme Linked Immunosorbent Assay (ELISA) for the Detection of Scedosporium/Lomentospora

Specific anti- S. boydii IgG was measured using a customized enzyme-linked immunosorbent assay (ELISA) method. ELISA was performed by coating wells of high binding micro test plates (Sarstedt, Nümbrecht, Germany) overnight at 4ºC with 10 µg/ml of protein extract diluted in sterilized PBS (100 µl per well). The following day the wells were washed three times with 200 µl PBS and blocked for 1 h at 37ºC by adding 200 µl of 5% (w/v) skimmed milk powder solution in PBS containing 0.05% (v/v) Tween 20 (PBST). Thereafter, three washes with PBST preceded the incubation at 37ºC for 1 h with 100 µl of human sera diluted 1:200 in PBST. In parallel, PBST without serum was added to some wells as negative control. Then these plates were washed three times with PBST, and 100 µl of HRP labeled anti-human-IgG (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:10,000 in PBST was added to each well. After 1 h incubation at 37ºC, three washes with PBS preceded the incubation with 50 µl TMB substrate solution (Thermo Fisher, Waltham, MA, USA) for 30 min in the dark at 24ºC. Finally, the reaction was stopped by adding 50 µl of 0.5 M H ₂SO ₄ and absorbance (Abs) was measured at 450 nm using a Synergy TM HT plate reader (BioTek, Winooski, VT, USA).

A pool made up of the five Scedo+ sera selected was included in each experiment as a positive control to monitor batch-to-batch variations and to normalize the data after the study.

Data Processing, Statistical Treatment, and Analysis

For data analysis, absorbance (Abs) values obtained in negative control wells were subtracted from remaining wells. Serum samples were measured in duplicate, and three replicates were performed for each ELISA experiment. With the aim of avoiding plate to plate bias variation, each Abs value was divided by the Abs value of the positive control included in all the plates and the result was expressed as Relative Abs.

Successive statistical analyses were run in SPSS Statistics software version 24 (IBM, Armonk, NY, USA) and the data plotted using Prism7 software (GraphPad, San Diego, CA, USA). Data distribution and its normality was studied by box plot analysis (see Supplementary Material 1). Two outlying values, classified into the Scedo- group, were identified and excluded from the analysis hereinafter. Normal distribution of data was detected by the Shapiro-Wilk test (< 50 samples) or the Kolmogorov-Smirnov test (> 50 samples), and homogeneity of the variance was proven by Levene test. Mean IgG response was compared between the three sera groups (Scedo+, Asp+, and Scedo-/Asp-) by performing a one-way analysis of variance (ANOVA) for normal distributed data or by Kruskal-Wallis for data with non-normal distribution, followed by Bonferroni's multiple comparison test. Likewise, Scedo+ group's mean specific IgG response was compared to the mean response of Scedosporium negative samples (Asp+ and Scedo-/Asp-) by performing Student's t test or the Mann-Whitney U test for data with a normal and non-normal distribution, respectively. All the analyses were performed taking into account a confidence interval (CI) of 95%, for this a p-value < 0.05 was considered statistically significant.

The optimal diagnostic cut-off value to discriminate positive and negative results was selected by taking into consideration the following seven criteria defined in the bibliography ( Habibzadeh et al., 2016; Mina et al., 2017; Unal, 2017): Youden Index ( J), Concordance Probability Method (CZ), Index of Union (IU), Closest to [0.1] criteria (ER), Control mean Abs plus 2 standard deviation (SD) , and Sensitivity = Specificity (SE = SP), and the maximum positive likelihood ratio (MLR+).

To calculate the cut-off value of Abs by some of these criteria an analysis of the Receiver Operating Characteristic (ROC) curve was performed, and the performance of the test was checked based on the area under the ROC curve (AUC). To do this, Abs obtained with each serum sample of Scedo+ group were considered as Patient Values, meanwhile Abs of sera from Asp+ and Scedo-/Asp- groups were included in Control Values. In this way, the ROC area under the curve (AUC) was drawn with 95% CI.

Finally, the accuracy and test performance of the ELISA was evaluated by comparison with the "Gold Standard" method (mycological culture of the sputa), calculating validation parameters of Sensitivity (SE), Specificity (SP), Positive Predictive Value (PPV), Negative Predictive Value (NPV), and Efficiency (EFF). In addition, agreement between the two techniques was analyzed by Cohen's Kappa index (K), which excludes the possibility of agreement occurring by chance.

Results

Fungal Prevalence in CF Patients' Sputum Samples

The epidemiological study of the group of sera used in this work, carried out according to the microbiological examination of patients' sputum (), showed that Candida yeasts were detected in 51.43% of the patients, C. albicans being the most frequently isolated (74.07%). Regarding filamentous fungi, 31.43% of the patients resulted in positive cultures for species in the genus Aspergillus, A. fumigatus being the most prevalent (66.67%), followed by Aspergillus terreus (39.39%) and species of Aspergillus flavus complex (3.03%). Meanwhile, 11.43% of CF patients were positive for Scedosporium/Lomentospora, with the highest impact from the S. apiospermum species complex (83.33%) and a significant prevalence of L. prolificans (50%). Likewise, 36.19% of the population studied showed negative cultures for these three fungal pathogens.

Reactivity of Human Sera Against S. boydii Protein Extracts

With the aim of analysing the immunoreactive capacity and the discriminatory power of S. boydii protein extracts, specific IgG reactivity of five sera from different patients selected from each of the three groups of CF sera were measured by ELISA using four different types of protein extracts as antigen: Total WCP, Secretome, Total CSP, and Conidial CSP (). In this sense, all extracts allowed the differentiation of the three groups, with the differences between Scedo+ and the other two groups, separately or together, statistically significant. From the four extracts, the best results were obtained with the Total WCP () as discrimination of sera was the most accurate, and there was no overlap between groups.

Although the secretome extract offered almost as accurate results, when evaluating the efficiency of the extraction methods () in terms of processing time, initial culture volume required, protein concentration, and volume of useful extract, the secretome extraction method was very time-consuming and yielded a lower concentration of protein.

IgGs Cross-Reactivity Study With Total WCP Extract of L. prolificans, A. fumigatus, and C. albicans

Selected sera were also tested against total WCP extract of the related species L. prolificans, and the most representative species of the two genera most frequently isolated in CF, A. fumigatus, and C. albicans (). ELISA values showed that there is a high cross-reactivity between groups of patients when the antigens of A. fumigatus and C. albicans are used. Meanwhile, L. prolificans extract was able to discriminate between groups, with the differences being statistically significant. This demonstrates that there is high cross-reactivity between Scedosporium and Lomentospora because protein extracts of both fungi succeeded in discriminating Scedo+ patients (). Moreover, sera from Scedo+ groups seemed to cross react with Aspergillus extract (), although the contrary was not observed, as sera from Asp + did not detect Scedo+ extract at the same level (Fig 5A). In the case of the C. albicans extract, sera from the three groups showed high reactivity against it ().

Total WCP Extract of S. Boydii as a Valuable Tool for Serological Detection of Scedosporium/Lomentospora

In order to evaluate S. boydii Total WCP extract as a serodiagnostic tool for Scedosporium/ Lomentospora detection, sera from the collection were tested individually using ELISA ().

When plotting all the values obtained in the ELISA assay, the three categories of sera were well distinguished. Indeed, differences between the median value of the specific IgG response for group Scedo+ compared to that obtained for Asp+ and Scedo-/Asp- were statistically significant, and also differed significantly from that obtained considering all Scedosporium negative samples together (Asp+ and Scedo-/Asp-) (***p < 0.001).

The diagnostic performance of the test was assessed by conducting an ROC analysis that showed an AUC value of 0.9942, meaning a high discrimination capacity (). Nevertheless, to assert whether a result was positive (detection of Scedosporium/ Lomentospora) or negative, a decision threshold had to be established, so seven different criteria were used: J, CZ, IU, ER, , SE = SP, MLR+ ().

Table 1

According to the above-mentioned criteria, four cut-off values were identified: Abs 450nm > 0.5837 using J, CZ, IU, and ER criteria, and Abs 450nm > 0.6042, > 0.6404 and > 0.7099 using SE = SP, , and MLR+, respectively. When evaluating the test performance, the SE was ≥ 86.9%, SP ≥ 93.6% and the PVN ≥ 98.4% regardless of the cut-off point selected. However, the PPV suffers a large variation, from 65%-95%, depending on the cut-off value. The K index was calculated as 0.76, 0.75, and 0.78 for the 0.5837, 0.6042, and 0.6404 cut-offs respectively, indicating a substantial agreement excluding chance. For the 0.7099 cut-off the K index calculated was 0.89 which indicates an almost perfect agreement. The cut-offs and the corresponding validation parameters are detailed in .

Monitoring of Specific IgG Levels Against Scedosporium

In order to determine the utility of the designed serological test, not only for serodiagnosis using a single sample point but also in the monitoring of CF patients, the Abs value of several serum samples corresponding to the same patient were plotted in a time-dependent manner according to the date of sample. The monitoring of patients (P) with at least three serum samples obtained over a period of more than 15 days is shown in and distributed in three different graphs according to the CF group in which they were previously classified. In this way, the evolution of the humoral IgG specific response against S. boydii can be observed and therefore, the evolution of the disease. In fact, some interesting trends where found when looking at the patients represented.

Scedo+ monitored patients () showed a high specific IgG response, which in all cases exceeded the cut-off values determined in this study. Specifically, P1 and P2 showed values that remained stable as clearly positive over the sampling interval. Conversely, P3 experienced an initial period of stability, but the final sample value decreased and crossed the threshold towards a negative outcome, and therefore, suggests a possible clinical improvement that should be confirmed with subsequent additional samples.

Regarding the Asp+ group () most of the serological profiles plotted corresponded to representative patients from the group, whose values were clearly under the diagnostic threshold. Nevertheless, two of the monitored patients showed a fairly high humoral response. P6 was close to the cut-off, even in certain sample times that crossed the threshold, while P5 exhibited a serological response that matches the profile of culture-confirmed Scedo+ patients. In this sense, these patients should remain under surveillance since they are at risk of presenting an undiagnosed Scedosporium colonization.

Finally, the monitoring of the patients from the Scedo-/Asp- group is illustrated in . Although most of the patients maintained negative results during the sampling period, P1, P2, and P3 showed intriguing serological profiles due to the high values detected. While P6 started with values close to the threshold but eventually developed a downward trend, P2 and P3 started the sampling with clearly negative results, however, high positive values were obtained in later samples. Which in turn, indicates that these patients, and especially P3, need to be carefully monitored since they may have an undetected presence of Scedosporium.

Discussion

The range of fungal species detected in CF patients continues to increase in line with new discoveries in diagnostic methodologies ( Tracy & Moss, 2018). The development of selective culture media has become a cornerstone for improving the classic gold standard of culture-based diagnosis, enhancing the recovery of low prevalence and/or slow growing fungal pathogens ( Pham et al., 2015; Hong et al., 2017; Coron et al., 2018). However, there are still huge limitations that hinder the correct detection of these fungi, such as the lack of standardized guidelines for processing respiratory samples and the absence of commercial available culture media designed for these challenging isolations ( Chen et al., 2017). On the other hand, serological tests have led to non-culture-based diagnosis of fungal infections since the 1950s because of their advantages, such as the ease of minimally invasive sample collection ( Richardson and Page, 2018). Unfortunately, at present, there is a lack of specific serological kits for low-prevalence fungal pathogens that enable rapid and easy detection.

For many years, efforts have been focused on Aspergillus fumigatus as it is the most prevalent filamentous fungi in CF airways ( Richardson & Page, 2017). In this sense, crude antigenic extracts from conidia and hyphae have been widely used for Aspergillus serological detection ( Page et al., 2016). On the other hand, species from Scedosporium genus are gaining more and more attention because of their high chronicity in CF airways as well as their associated pathogenicity. However, despite their disturbing clinical relevance, accurate detection methods that allow an adequate diagnosis of these threatening pathogens is currently lacking ( Chen et al., 2017). Mycological culturing continues to be the most widespread method. This requires the use of selective media that are not available everywhere, and incubation times for these slow-growers are excessively long ( Coron et al., 2018). This problem is reflected in the epidemiological studies that suffer worrying variations in the prevalence rates, caused to some extent, by the use of different and non-standardized procedures. In fact, there is a growing awareness of this issue and this has led to the publication of research into the frequency variation depending on the detection method employed ( Borman et al., 2010; Sedlacek et al., 2015; Hong et al., 2017; Boyle et al., 2018; Hedayati et al., 2019). These diagnostic hurdles in turn result in delayed diagnosis, and consequently, a late introduction of an effective treatment. Therefore, considering the mounting concern about the critical need for standardized and reliable detection methods, efforts must be made to develop rapid and robust tests to ensure an early detection of Scedosporium, and consequently the establishment of an effective treatment and patient monitoring. With this in mind, the aim of this study was to analyse the utility of different protein extracts of S. boydii for a serological detection of Scedosporium/ Lomentospora in CF patients by ELISA.

To achieve this, 212 serum samples corresponding to 105 CF patients were used. Patients were classified into three groups, Scedo+, Asp+, and Scedo-/Asp-, based on the fungus isolated from the sputum, Scedosporium, Aspergillus, or neither, respectively. Mycological cultures showed that Candida was the most frequently isolated fungus among yeasts, and Aspergillus among filamentous fungi. Nevertheless, Scedosporium species were isolated from 11.43% of CF patients' sputa, these results were in agreement with the prevalence rates of 8%-16% published in different epidemiological studies ( Cimon et al., 2000; Blyth et al., 2010; Schwarz et al., 2017). Moreover, species of S. apiospermum complex were isolated in 85.33% of Scedo+ patients, which is consistent with the Scedosporium species distribution in CF, since S. apiospermum and S.boydii are the most frequently isolated species ( Bouchara et al., 2019). Finally, L. prolificans was detected in 50% of Scedo+ patients, which is a notoriously high prevalence rate when compared with the worldwide incidence data (0%-40%), but it is in concert with the geographical restriction of the fungus, Spain and Australia being the countries with the highest incidence ( Seidel et al., 2019).

The first step in the design of the ELISA for the serological detection of Scesdosporium/Lomentospora was the selection of an easy-to-obtain protein extract with a good level of discrimination. Some research has deciphered potential Scedosporium virulence markers (reviewed in Santos et al., 2009) with different cellular locations that are related to both morphological phases (hypha and conidium). Bearing in mind the wide variety of antigens, the immunoreactivity and discriminatory capacity of four S. boydii protein extracts, which included total protein extract (Total WCP), secretome extract, cell surface associated proteins (Total CSP), and cell surface associated proteins only from conidia (Conidia CSP), were compared by indirect ELISA against fifteen patients (five from each CF group). The results obtained with the four extracts showed that they were able to discriminate Scedo+ sera from Asp+ and Scedo-/Asp- groups. However, of the four extracts, Total WCP was selected to continue with the study as it was the one with the highest output in terms of protein concentration per mL of fungal culture used, and the discriminatory capacity was the best because none of the five patients tested in the Scedo+ group overlapped with any of the other two groups.

Among the discarded extracts, secretome was expected to be interesting because of the chronicity of Scedosporium colonizations of CF airways and the specificity of the secreted proteins according to bibliography ( Santos et al., 2009; Bertrand et al., 2010). Moreover, metalloproteases such as superoxide dismutase, some proteolytic enzymes, as well as ectophosphatases are secreted to the external environment and play a protective role for the fungus and orchestrate the cleavage of key host components ( Larcher et al., 1996; Da Silva et al., 2006; Da Silva et al., 2012). In agreement with this idea, the results obtained using this extract were very good, but similar to the ones obtained with the Total WCP and, additionally, there were some problems with gathering high protein concentrations, a long and complex process was necessary to obtain an extract with low yields.

Regarding Total CSP and Conidial CSP, it is well known that some cell wall associated proteins of Scedosporium, such as glucans, peptidorhamnomanns and glucosylceramides, show a high immunoreactive capacity ( Pinto et al., 2004; Da Silva Xisto et al., 2019). Moreover, since the typical entry of the fungus into the host body is through inhalation of conidia, it is reasonable for proteins of the cell wall of the conidium to play an important role in the host-pathogen interactions and colonization/infection of airways ( Buldain et al., 2016). Consequently, as with the secretome, the results obtained were also good but yielded low protein concentration and in addition, some overlapping between groups with the Conidial CSP was observed. This cross-reactivity could be explained by the fact that some surface proteins of the conidia are common to different moulds, are important for the survival of the fungus, and possess immune-modulatory functions ( Voltersen et al., 2018).

Hence, further experiments were carried out using Total WCP. Crude antigenic extracts have been widely used in immunodiagnostic systems, some of them being currently commercially available for the detection of Aspergillus ( Page et al., 2016; Richardson and Page, 2017). The advantage of these kinds of extract is that they involve every interesting antigen. On the contrary, one of their main handicaps is their cross-reactivity against panfungal antigens of closely related fungi ( Buldain et al., 2019). In this sense, biochemical studies try to characterize specific proteins for diagnostic purposes, but as little is known about the physiology and biochemistry of Scedosporium, and the number of studies performed in the field is small, only a few proteins of interest have been characterized. Among these are a serine protease from the subtilisin family, two enzymes involved in ROX detoxification, cytosolic Cu,Zn-superoxide dismutase, a catalase and some heat shock proteins ( Santos et al., 2009).

Moreover, the potential of crude antigenic extracts of Scedosporium/Lomentospora, which is the first step in the serodiagnosis race, has not yet been evaluated. In this way, Total WCP was tested against the totality of sera by the same optimized ELISA assay, and the results show that it was able to discriminate the Scedo+ patients successfully. In this sense, ROC AUC analysis possesses a good discriminatory capacity for the test (AUC=0.9942) but assessing the criteria for cut-off determination ( Habibzadeh et al., 2016; Unal, 2017), four threshold values were represented. The most affected parameter was the theoretical PPV*, because a PPV* of 65% was obtained with the lowest threshold values but it increased to 95% in the highest one. Theoretical NPV*, efficiency and specificity values remained more or less stable in all the cases, while sensitivity varied from 86%-100% with the highest and the lowest cut-offs, respectively. Despite the variations observed, the ELISA assay showed a remarkably high specificity, which means an absolute capacity to discriminate CF patients with no presence of Scedosporium. Moreover, it was observed that being stricter with the selection of the cut-off value resulted in improved ability to detect Scedosporium positive patients. Nevertheless, choosing one cut-off value may be risky considering the limited number of Scedo+ samples. In this sense, increasing this sample population might be helpful to establish a definitive cut-off point.

Turning to the commercial kits for Aspergillus detection that show a 75%-96% sensitivity ( Richardson and Page, 2017), the developed test exhibits a remarkable clinical usefulness considering that values of 86 - 100% of sensitivity and 93%-99% of specificity were obtained. Moreover, it is worth mentioning that Lomentospora- positive culture patients also showed a specific response against S. boydii extract, so the developed ELISA platform is a valuable tool to detect the intrinsic multi-resistant fungus L. prolificans as well. In the last few years, other authors have made efforts to shed light on serodiagnosis of Scedosporium. In fact, Bouchara and co-workers in 2017 developed an ELISA with two recombinant proteins, Scedosporium catalase A1 and cytosolic Cu,Zn-superoxyde dismutase, described as antigens with diagnostic utility in Aspergillus. In this study, they managed to detect Scedosporium infections, and differentiate it from an Aspergillus infection. Moreover, precipitin assays can be performed but they take up to one week for the results to be obtained, lack sensitivity ( Fujiuchi et al., 2016), and can only be performed in a few specialized laboratories ( Cimon et al., 2000).

Finally, the study of the ELISA test as a patients' monitoring tool showed the value of the technique for the observation of the evolution of the fungal presence since the humoral response of the patient can be tracked over time, although this point should be studied in more depth in the future. Nevertheless, the best diagnosis is the variety of tests with different purposes that complement each other and offer a real vision of the patient's condition and evolution. In this sense, our research group aimed to explore new diagnostic resources by developing an indirect ELISA test using S. boydii whole cell protein extract. Detecting humoral response against Scedosporium in CF patients regardless the result of the culture may be helpful for clinicians to maintain these patients under surveillance, and to anticipate the establishment of their antifungal treatment.

Conclusions

In this study a crude antigenic extract of S. boydii was selected to detect Scedosporium/Lomentospora serologically. The ELISA test developed is able to detect the Scedosporium spp. and L. prolificans in CF patients' sera, with a very high sensitivity and specificity, up to 100% and 99%, respectively. Thus, this ELISA platform offers a rapid, low-cost and easy solution to detect these elusive fungi through minimally invasive sampling, with high output and specificity, and allows the monitoring of the evolution of the infection, the recovery and the effectiveness of the antifungal therapy. In spite of the potential of these results, more research is needed in this field to detect specific antigens from the fungal extract to improve sensitivity and specificity, to minimize cross-reactivity with other closely related fungal pathogens, and ultimately to reach a standardized method. Nevertheless, it is worth bearing in mind that the best diagnosis is achieved with a combination of methods that allow a complete vision of the infection.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics Statement

The studies involving human participants were reviewed and approved by Ethics Committee from the UPV/EHU. The patients/participants provided their written informed consent to participate in this study.

Author Contributions

LM-S, IB, LA-F, and MA carried out the experiments. J-PB and MM-G obtained and classified the sera samples. AR, FH and AR-G conceived the experiments and supervised the work. LM-S, AR, FH, and AR-G analyzed the data. LM-S and AR-G wrote the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This research was funded by the Basque Government, grant number IT1362-19. IB, LM-S, and LA-F received a predoctoral fellowship from the Basque Government. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We thank the Member of the Chartered Institute of Linguists number 022913 for improving the English.

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# data[].attributes.attachment_link.title
ELISA Test for the Serological Detection of Scedosporium/Lomentospora in Cystic Fibrosis Patients

# data[].attributes.attachment_link.description
The detection and diagnosis of the opportunistic fungi Scedosporium spp. and Lomentospora prolificans still relies mainly on low-sensitive culture-based methods.

# data[].attributes.headline
Standardization of ELISA protocols for serosurveys of the SARS-CoV-2 pandemic using clinical and at-home blood sampling. 

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Standardization of ELISA protocols for serosurveys of the SARS-CoV-2 pandemic using clinical and at-home blood sampling. 

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Klumpp-Thomas, Carleen, Heather Kalish, Matthew Drew, Sally Hunsberger, Kelly Snead, Michael P. Fay, and Jennifer Mehalko et al. 2021. "Standardization Of ELISA Protocols For Serosurveys Of The SARS-Cov-2 Pandemic Using Clinical And At-Home Blood Sampling". Nature Communications 12 (1). doi:10.1038/s41467-020-20383-x.

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doi: 10.1038/s41467-020-20383-x.

Affiliations

¹ National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, 20850, USA.

² Section on Immuno-Engineering, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, 20894, USA.

³ Trans-NIH Shared Resource on Biomedical Engineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, 20894, USA.

⁴ Protein Expression Laboratory, NCI RAS Initiative, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA.

⁵ Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20894, USA.

⁶ LID Clinical Studies Unit, Laboratory of Infectious Diseases, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20894, USA.

⁷ Section on Immuno-Engineering, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, 20894, USA. kaitlyn.sadtler@nih.gov.

Free PMC article

Free PMC article

doi: 10.1038/s41467-020-20383-x.

Affiliations

¹ National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, 20850, USA.

² Section on Immuno-Engineering, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, 20894, USA.

³ Trans-NIH Shared Resource on Biomedical Engineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, 20894, USA.

⁴ Protein Expression Laboratory, NCI RAS Initiative, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA.

⁵ Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20894, USA.

⁶ LID Clinical Studies Unit, Laboratory of Infectious Diseases, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20894, USA.

⁷ Section on Immuno-Engineering, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, 20894, USA. kaitlyn.sadtler@nih.gov.

Abstract

The extent of SARS-CoV-2 infection throughout the United States population is currently unknown. High quality serology is key to avoiding medically costly diagnostic errors, as well as to assuring properly informed public health decisions. Here, we present an optimized ELISA-based serology protocol, from antigen production to data analyses, that helps define thresholds for IgG and IgM seropositivity with high specificities. Validation of this protocol is performed using traditionally collected serum as well as dried blood on mail-in blood sampling kits. Archival (pre-2019) samples are used as negative controls, and convalescent, PCR-diagnosed COVID-19 patient samples serve as positive controls. Using this protocol, minimal cross-reactivity is observed for the spike proteins of MERS, SARS1, OC43 and HKU1 viruses, and no cross reactivity is observed with anti-influenza A H1N1 HAI. Our protocol may thus help provide standardized, population-based data on the extent of SARS-CoV-2 seropositivity, immunity and infection.

Conflict of interest statement

The authors declare no competing interests.

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covid-19 serological testing / standards

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enzyme-linked immunosorbent assay / methods*

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enzyme-linked immunosorbent assay / standards*

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national center for biotechnology information

# data[].attributes.attachment_link.title
Standardization of ELISA protocols for serosurveys of the SARS-CoV-2 pandemic using clinical and at-home blood sampling - PubMed

# data[].attributes.attachment_link.description
The extent of SARS-CoV-2 infection throughout the United States population is currently unknown. High quality serology is key to avoiding medically costly diagnostic errors, as well as to assuring properly informed public health decisions. Here, we present an optimized ELISA-based serology protocol, ...

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Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization - Plant Methods

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Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization - Plant Methods

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Haring, Max, Sascha Offermann, Tanja Danker, Ina Horst, Christoph Peterhansel, and Maike Stam. 2007. "Chromatin Immunoprecipitation: Optimization, Quantitative Analysis And Data Normalization". Plant Methods 3 (1). doi:10.1186/1746-4811-3-11.

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Chromatin immunoprecipitation experiments can roughly be divided into two categories. One uses crosslinked chromatin sheared by sonication (X-ChIP), and the other native chromatin digested by nucleases (N-ChIP). Both methods have their own advantages and disadvantages [ 8]. Our paper provides and discusses a protocol for X-ChIP that involves formaldehyde crosslinking of the chromatin in intact tissue, ensuring the rapid fixation of the existing chromatin structure. Without crosslinking, we systematically failed to obtain significant amounts of precipitate. In this paper, we discuss the whole procedure in four sections: (1) The isolation of good-quality chromatin from plant material, (2) the chromatin immunoprecipitation itself, (3) the analysis of precipitated material by QPCR and (4) data normalization. An outline of the various steps in the whole procedure is shown in Figure 1.

(1) Isolation of chromatin

Starting material

The isolation of plant chromatin needs a plant-specific approach; plant cells are surrounded by a cell wall, and generally whole tissues, rather than uniform cell cultures, are used to isolate chromatin. Plant cells contain large vacuoles, resulting in a relatively low number of nuclei per gram of tissue. In addition, vacuoles are a source of proteolytic activities [ 9]. We advise the use of healthy, unfrozen plant tissue as starting material for the isolation of chromatin. If possible, use tissue enriched in unexpanded cells. Such tissue will provide the best yield and purity of the isolated chromatin. In case there are doubts if the tissue of interest will yield good-quality chromatin, non-crosslinked nuclei can be isolated followed by micrococcal nuclease digestion. Good-quality chromatin gives rise to a distinct nucleosome ladder [ 8, 10].

Crosslinking

Crosslinking of the starting material by formaldehyde is used to ensure that the chromatin structure is preserved during the isolation and ChIP procedure [ 6, 11]. Crosslinking of chromatin within the plant tissue requires the fixative to penetrate the cells, which is hampered by the plant's waxy cuticle and spongy air-filled mesophyll. In our protocol, efficient penetration of the fixative is achieved by vacuum infiltration of buffer that contains formaldehyde. It is important that the buffer penetrates the plant material completely; after vacuum infiltration the plant material should appear translucent or 'water-soaked'. The buffer volumes used in the presented protocol are suitable for efficiently crosslinking up to 5 grams of plant material. Increasing the amount of material impedes efficient crosslinking.

The crosslinking step should be optimized, since too little crosslinking will not sufficiently preserve the chromatin structure, and too much crosslinking will hamper the ChIP procedure [ 12]. A method to determine the optimal crosslinking conditions is illustrated in Figure 2. Essentially, when the crosslinking is optimal for ChIP, decrosslinking is required to efficiently isolate DNA from the nuclei by phenol-chloroform extraction. The chromatin is over-crosslinked when it is impossible to recover a substantial amount of DNA from the nuclei by decrosslinking. The nuclei are under-crosslinked when most of the DNA can be recovered without decrosslinking. Crosslinked material can be stored for several months at -80°C after freezing in liquid nitrogen.

Crosslinking efficiency analysis. Leaf material was crosslinked in buffer containing increasing amounts of formaldehyde. Samples were decrosslinked (+DC) or not (-DC), and DNA isolated using phenol/chloroform extraction followed by ethanol precipitation. While DNA is efficiently isolated from samples that were not crosslinked (lanes indicated with 0×), decrosslinking is required for the isolation of DNA from crosslinked samples (lanes indicated with 1× and 3×). Over-crosslinking strongly decreases DNA isolation efficiency (lanes indicated with 3×). DC, decrosslinking; 0×, 1×, 3×, relative concentration of formaldehyde; M, lambda DNA cut with Pst I.

Shearing of chromatin

The resolution obtained by the ChIP procedure is determined by the size of the chromatin fragments used as input material. Two methods are commonly used to fragment chromatin, sonication (hydrodynamic shearing) and micrococcal nuclease (MNase) digestion. Both methods can show preferential fragmentation of certain chromosomal regions [ 13, 14]. When using formaldehyde crosslinking, sonication is the preferred method, as crosslinking restricts the access of MNase to chromatin [ 15].

Optimal fragmentation can be achieved by testing various sonication conditions on chromatin, followed by DNA isolation and estimating the sonication efficiency by gel electrophoresis. Ideally, the bulk of the chromatin is sonicated to a length between 250 and 750 bp. For efficient fragmentation, sonication at low power, in combination with several pulses, is preferred over sonication at high power and few pulses, but conditions vary with the sonication device used. It is important to keep the chromatin sample cooled on ice during sonication, as heat released by the sonication probe can reverse the crosslinks. The presence of detergent (SDS) in the sonication buffer improves sonication efficiency considerably, but can induce foaming during sonication. Foam makes the chromatin sample unsuitable for ChIP, probably as a result of the surface tension imposed by the foam, which can disrupt protein conformation [ 16]. Foaming can be prevented by decreasing the sonication power. Sonicated chromatin can be stored at -80°C for at least a few months, but repeated freeze/thaw cycles should be avoided.

(2) The chromatin immunoprecipitation procedure

Choosing antibodies

Antibodies (Ab) are the most important factor for a successful ChIP experiment. It is crucial to choose an antibody carefully, especially when antibodies are raised against non-plant proteins. Papers reporting the use of a specific antibody for ChIP are a good indication of the suitability of that antibody, but one should realize that quality can differ between antibody batches. The successful use of a specific antibody in experiments other than ChIP (i.e. Western blotting, immunocytochemistry) does not automatically mean the antibody is suitable for ChIP; that has to be tested.

Antibodies can be available as polyclonal or monoclonal preparations. Monoclonal antibodies have a high specificity compared to polyclonal sera, but the polyclonal sera may recognize several epitopes of the target, increasing signal levels of low-abundance templates. Be aware that polyclonal serum against multiple modifications (i.e. against hyperacetylated H4) may have an undocumented preference for one modification over the others [ 17], impeding the biological interpretation of ChIP data.

Different antibody preparations have distinct properties, which can affect the ChIP results. The affinity for epitopes differs between antibodies, affecting the resulting signal levels. For example, antibodies can differ in their sensitivity towards crosslinks or adjacent modifications (discussed in [ 18]). In addition, the relation between the availability of epitopes and antibody binding may not be linear. Some antibodies are sensitive to inhibitory factors present in the input chromatin sample, resulting in a decrease in binding efficiency of the antibody when increasing the amount of input. This is exemplified in Figure 3. We tested the effect of changing the amount of input chromatin on ChIP efficiency (the recovery of DNA relative to the amount of input) for two different antibodies. With an antibody against hyperacetylated H4 (Upstate #06-946), the ChIP efficiency is constant over a broad range of chromatin concentrations. When this experiment is performed with an antibody recognizing an invariant domain of H3 (H3core; Abcam #AB1791), dilution of the input chromatin improves the ChIP efficiency. These data show that the relation between ChIP signal and chromatin input depends on the characteristics of the antibody used. To ensure the comparability of results obtained with different input samples, we recommend determining the optimal Ab: chromatin ratio by titration of the amount of input chromatin, and the use of similar amounts of input chromatin when ChIP results are to be compared.

Titration of chromatin relative to a fixed amount of antibody. (a) ChIP titration experiment using an antibody against acetylated H4. Dilution of the chromatin has no effect on the precipitation efficiency. (b) ChIP titration experiment using an antibody against an invariant domain of H3 (H3core). Dilution of the chromatin improves the precipitation efficiency, suggesting that the antibody recognizing the H3core is sensitive to inhibitory factors present in the chromatin sample. ChIP-QPCR was performed as described in this paper. Chromatin was isolated from leaves of 2 week-old plants, sonicated and used in 1:1, 1:2 and 1:4 dilutions for ChIP. The input samples are diluted by the same factors as the chromatin samples. Results are represented as %IP, the error bars indicate the standard error. ChIP samples are represented by closed bars, while open bars indicate the signals from the 'no-antibody' serum controls. Each data point represents the average of two different chromatin samples, each analyzed in duplo.

When establishing the ChIP procedure we recommend using an antibody against an invariant domain of histones as a starting point [ 19, 20]. In our hands, such an antibody usually results in a good signal-to-noise ratio. When setting up ChIP to detect histone acetylation, the initial detection can be facilitated by treating plant material with butyrate or TSA before the isolation of chromatin [ 7, 20]. Such treatments inhibit histone deacetylation, resulting in increased global acetylation levels.

Once the ChIP protocol has been established, it has to be determined which histone modifications will be analyzed. For this purpose, data obtained from immunocytochemistry experiments are a valuable source of information [ 21, 22]. Such experiments should however, be considered as informative but not conclusive for the decision on which histone modification to analyze. Immunocytochemistry experiments provide a global view of the localization of histone modifications at eu- or heterochromatin, while ChIP experiments can have a single-nucleosome resolution [ 23]. At such high resolution, histone modifications can be observed where they are not expected based on immunolocalization experiments ([ 20, 22]; Haring et al, in preparation). Another factor to keep in mind is that specific chromatin states can be associated with different histone modifications in distinct species. For example, immunolocalization experiments showed the presence of H3K9me2, but not H3K9me3, in heterochromatin of Arabidopsis, while H3K9me3, but not H3K9me2, is observed in heterochromatin of mice [ 21, 24, 25].

Controls for ChIP

To ensure the reliability of ChIP data, two control samples specific for the ChIP experiment should be included: the input sample and the 'no-antibody' (NoAb) control sample (Table 1). Both the input sample and the NoAb control sample provide essential information about the ChIP experiment, and should be analyzed with every primer set used. Additional controls are needed for the QPCR procedure; these will be discussed in the QPCR section.

The input sample will be indicative for the presence and amount of chromatin used in the ChIP reaction. It is an aliquot taken from the chromatin before preclearing (step 9 in the protocol). The chromatin aliquot is decrosslinked and DNA is isolated. This DNA sample should yield a PCR product with all primer sets used. Besides serving as a positive control, the data derived from the input sample can be used for normalization by the '%IP' method discussed in the section on data normalization.

The NoAb control is a chromatin sample to which non-specific control serum is added instead of a specific antibody (see materials and methods). The NoAb sample is treated the same way as the ChIP samples. The QPCR signals resulting from the NoAb samples indicate the amount of background signal generated by the chromatin preparations and ChIP procedure. Ideally, the washing steps remove non-specifically bound chromatin, resulting in an absence of QPCR signals for the NoAb samples. In reality however, it is not uncommon to find a PCR product for the NoAb control sample. It is very important that the DNA isolated from the NoAb samples is amplified with every primer pair used, as the level of background signal can differ for each primer pair.

Optimizing the signal to noise ratio

When setting up ChIP, one of the main problems is a high level of background signal (NoAb control) relative to the level of the signal of interest (ChIP sample). This hampers distinguishing a 'true signal' from the background signal. Therefore, we recommend setting up ChIP using an antibody against an invariant domain of histone H3 [ 19, 20]. In our experiments, such an antibody yields a very clear difference in signal between the ChIP samples and NoAb control. Another helpful tool to discriminate between background and ChIP signal is a positive control sequence for ChIP, as discussed in the 'controls for specific chromatin states' section.

Several options are available to optimize the signal-to-noise ratio. To prevent non-specific binding of chromatin to the protein A/G agarose beads, the beads are blocked with BSA and non-specific blocking DNA before they are used in ChIP assays. To further reduce background, BSA, non-specific serum and unrelated DNA can be added to the pre-incubation step of chromatin with beads (step 9). Options for preventing chromatin from binding to plastic tubes are pre-incubation of the tubes with BSA and unrelated DNA (e.g. salmon sperm DNA), or the use of siliconized tubes. The background can also be reduced by lowering the amount of input chromatin, the amount of protein A/G agarose beads or the amount of antibody, but this may lower signal levels as well.

In case ChIP experiments with an antibody against an invariant domain of histone H3 would result in a low ChIP-QPCR signal relative to the background signal, factors inhibiting the PCR reaction may be involved. We recommend purifying the DNA after the reverse crosslinking (step 17) with a commercial 'PCR purification spin column kit'. In our hands, DNA purification by phenol-chloroform extraction does not sufficiently remove detergents present in the ChIP elution buffer, inhibiting subsequent QPCR reactions.

(3) Analysis of precipitated material by QPCR

DNA isolated from the precipitated chromatin has to be analyzed to determine which DNA fragments are present in the precipitate (Figure 1). For the detection of specific DNA fragments, various methods are available, and the chosen method determines to what extent the data can be analyzed quantitatively. Commonly used analysis methods are conventional PCR and quantitative PCR. Alternative methods are microarray analysis and slot blotting. Microarray analysis is useful when studying the genome-wide distribution of histone modifications [ 26]. Analysis by slot blotting is feasible when dealing with highly repetitive sequences [ 27, 28], but appears not sensitive enough to detect single copy sequences in ChIP samples (M. Haring and M. Stam, unpublished). This paper focuses on quantitative PCR for the detection of immunoprecipitated DNA (see Figure 4 for an outline how to set up QPCR).

Outline for setting up QPCR. This outline represents setting up the QPCR analysis, as described in the text. Abbreviations: TF: transcription factor.

Until recently, ChIP precipitates have primarily been analyzed by conventional PCR (e.g. bands on a gel). Such an approach requires lots of testing per individual primer set to ensure that measurements are taken in the linear range of amplification. If this condition is not fulfilled, the resulting data cannot be considered quantitative, impeding data interpretation. For this reason, analyzing ChIP precipitates by quantitative real-time PCR (QPCR) has several advantages over conventional PCR. The QPCR technique does not quantify the amount of PCR product at the end of the PCR reaction, as with conventional PCR band densitometry. Instead, the initial amount of template DNA is calculated from the kinetics of the PCR reaction. During a QPCR run, the accumulation of PCR product is measured every cycle. The number of cycles needed to reach a certain amount of PCR product - 'Cycle threshold' or Ct value [ 29]-, and a calibration line (see below), are used to calculate the initial amount of DNA template.

Quantitative PCR analysis for ChIP can be performed using DNA-dye based or probe-based PCR product detection chemistries. The most widely used DNA-dye based QPCR chemistry employs the fluorescent dye SYBRgreen for detection of the amplicon. SYBRgreen is only fluorescent when bound to double-stranded DNA (dsDNA), and the amount of fluorescence is proportional to the amount of dsDNA. SYBRgreen detects a PCR product independent of its DNA sequence, so this chemistry can be used for all primer sets. At the same time, this sequence-independent binding of SYBRgreen requires primer sets to be thoroughly optimized; amplification of non-target DNA fragments and formation of primer dimers will also yield fluorescent signal, hampering measurements of the ChIP-QPCR signal.

When using hybridization probe-based chemistries, one or more fluorescently labeled oligonucleotide probes are designed to anneal to a specific DNA sequence [ 29]. This adds specificity to the detection of PCR fragments, as both the primers and the probe will have to anneal to template DNA to enable detection of the product.

QPCR primer sets

In order to obtain high quality QPCR data, the primer sets used need to meet specific criteria [ 29, 30]. If primer sets are not optimized, this may result in amplification artifacts and/or an inaccurate quantification. Primer sets can be tested by performing QPCR on serial dilutions of template DNA isolated from crosslinked, sonicated chromatin. The efficiency of the amplification should be as close as possible to two (a 2× dilution of the template should result in a 1 Ct increase), and should remain constant over a wide range of template concentrations. The amplification efficiency can be improved considerably by designing the length of the amplified fragment between 80 and 150 bp. Importantly, the ChIP-QPCR results should be within the linear range of amplification. To verify that the primer sets amplify the correct fragment, sequencing the obtained PCR product is recommended. When using SYBRgreen for detection of the QPCR signal, the amplification of non-target DNA and the formation of primer dimers should be avoided, as the dye does not discriminate between these and target DNA fragments (discussed above).

The sequence regions to be amplified have to be chosen very carefully. Different combinations of histone modifications mark distinct DNA sequence elements, such as regulatory sequences, promoter- or coding sequences. The histone modifications present at such specific DNA elements can be different from those at the flanking chromatin [ 31]. We therefore advise to design primer sets within a single DNA element, such as a promoter, intron or other regulatory sequence. When the amplified region spans more than one DNA element, the data obtained by ChIP may consist of a mixture of data, hampering the biological interpretation. In conclusion, knowing as many features of the amplified sequences as possible is useful for the design of useful primer sets.

Controls for QPCR

A good-quality QPCR analysis can only be performed when taking along the proper controls. These controls are in addition to the earlier discussed input and NoAb controls, which are specific for the ChIP experiment. This section will discuss the required QPCR specific controls: a calibration line, melting curve analysis and the no-template control (Table 1B).

A QPCR calibration line is required for the quantification of ChIP signals, and should be included for every primer set used. A calibration line consists of QPCR reactions performed with multiple dilutions of template DNA that is isolated from sonicated crosslinked chromatin. The calibration line corrects for differences in PCR efficiency between various primer sets, as well as for differences in signal level between multicopy and single copy sequences. Besides enabling quantification, the calibration line functions as a positive control for the QPCR assay, independent of the ChIP samples. The dilution series for the calibration line must be made fresh from a DNA stock every time, as the quality of diluted DNA rapidly decreases. The latter is probably due to binding of DNA to the surface of the reaction tube [ 32]. Addition of BSA or unrelated DNA may reduce this effect.

When performing QPCR with SYBRgreen, a melting curve analysis can be performed to determine if the correct fragment is amplified. In this extension of the PCR program, the temperature is gradually increased while continuously measuring SYBRgreen fluorescence. PCR products will denature at a temperature specific for their size and sequence, and this will be measured as a loss of fluorescence. Multiple PCR fragments in one reaction, or the presence of primer-artifacts, will result in a step-wise decrease of fluorescence. Optimal primer sets should yield a single denaturation event. Even so, multiple fragments of similar size and/or sequence may yield identical melting curves. The PCR products should therefore be verified by sequencing before using the primers for ChIP-QPCR analysis. The melting curve analysis is not possible when using QPCR chemistries based on fluorescent oligonucleotide probes.

A no-template control, which is commonly included in conventional PCR, is useful for the detection of master mix contamination and primer dimers. The no-template control is distinct from the NoAb control, as the latter usually results in PCR products.

(4) Controls for specific chromatin states

Genomic sequences which are known to be associated with specific histone modifications are essential controls for the interpretation of ChIP data (Table 1C). When, in a ChIP experiment, the regions of interest do not show any enrichment of a specific histone modification, the control sequences can indicate if this is due to a failed experiment or if the outcome reflects the actual situation. Ideally, the controls function as a positive and negative control for several antibodies in various tissues.

The development of positive ChIP controls is challenging as chromatin states can not simply be classified as "active" or "silent", many different types of active and silent chromatin exist. These chromatin states are the result of several different molecular networks that can act together at a particular genomic location, each resulting in a distinct set of histone modifications and other chromatin features [ 33, 34]. Identifying a positive control for a specific chromatin state is facilitated by knowledge of the regulatory processes that underlie the presence of the histone modification of interest.

Here, we will discuss controls for two different chromatin states: transcriptional active and repressed chromatin. In our example, the positive control for active chromatin is a negative control for repressed chromatin and vice versa.

Active chromatin

Importantly, if antibodies are used against histone modifications associated with active chromatin, the control sequence for active chromatin must result in a positive signal in all tissues of interest. A good candidate is a housekeeping gene that is constitutively expressed, such as the actin gene, which is frequently used as a positive control for active chromatin [ 35- 39]. Even if the expression level of a control gene varies in different tissues, it may still function as a good control; quantitative changes in expression level do not necessarily reflect changes in histone modifications.

We have used maize actin1 coding sequences to develop positive controls for active chromatin. Cloning and sequencing of actin1 DNA from our maize lines indicated it is a single copy gene and well suited for QPCR analysis. RT-PCR analyses showed it is active in the tissues we have examined (husk and young leaves; data not shown). ChIP was performed as described in this paper, and input chromatin was isolated from leaves of one month old plants (young leaves) and from husks of three months old plants (husk leaves). The precipitates were analyzed with primers specific for two positions on the actin gene: the untranslated leader (UTR) and the second exon (exon 2; Figure 5a). We observed positive signals for both the actin UTR (Figure 5b) and exon 2 (Figure 5c) when using antibodies against H3K4me2, H3K9ac/K14ac (H3ac), H4ac, but no signals above background with antibodies against H3K9me2 and H3K27me2 (Figure 5 and data not shown). These data show that this sequence can be used as a positive ChIP control for histone modifications associated with active chromatin, and a negative control for histone modifications correlated with repressed chromatin.

ChIP analysis of control sequences. (a) Location of amplicons used in ChIP-QPCR analysis. The boxed regions indicate part of the coding sequence of maize actin 1 (genbank #J01238) and the reverse transcriptase sequence of the maize copia TY1 type retrotransposon (genbank #AF398212). The amplified sequences are indicated by bars. (b) ChIP-QPCR analysis of the actin 5' untranslated leader (UTR). (c) ChIP-QPCR analysis of the actin exon 2 fragment. (d) ChIP-QPCR analysis of the copia sequence ( copia). ChIP-QPCR is performed as described in this paper. Input chromatin was isolated from leaves of 4 weeks old plants (young leaves) and from husks of 3 months old plants (husk leaves). The ChIP results obtained by 4 independent replicate experiments are represented as percentage of input (%IP), the error bars indicate the standard error. The ChIP signals are represented by closed bars, and open bars indicate the signals from the no-antibody control (NoAb).

Our data in addition demonstrate the importance of carefully selecting the regions to test in ChIP-QPCR (see also the QPCR primer sets section). In young leaves, the actin exon 2 carries a low level of H3K4me2 (figure 5b), while the actin UTR carries a significantly higher level of H3K4me2 (Figure 5c; 95% confidence interval). When performing a ChIP experiment with an antibody against an invariant domain of H3 (H3core), the signals observed for actin UTR and exon 2 were comparable, indicating that the difference in H3K4me2 signal levels between UTR and exon 2 is not due to a difference in nucleosome density, but instead reflects a genuine difference in the H3K4me2 levels. This difference is specific for H3K4me2, as the H3ac signal levels are not significantly different between actin UTR and exon 2 in young leaves. The observed differences in signal levels of H3K4me2 may reflect differences in the molecular mechanisms acting on the distinct sequence elements of the actin gene.

Repressed chromatin

To develop a positive control for repressed chromatin, transposon sequences may be considered. Different types of transposons exist, varying in copy number, DNA sequence features and susceptibility to different forms of epigenetic regulation [ 40, 41]. The vast majority of the highly repetitive transposon sequences within plant genomes are truncated [ 42]. This makes them unfavourable templates for QPCR, because amplification of truncated sequences results in a range of PCR products, impeding correct quantification. For maize, we successfully developed a control for repressed chromatin using the highly conserved TY1 class copia LTR retrotransposon reverse transcriptase sequence [ 43]. The expression of a copia-like transposon was shown to be repressed in rice [ 44], and RT-PCR experiments suggested it is in maize as well (data not shown). We examined the presence of histone modifications at the copia sequence (Figure 5a) using the same ChIP precipitates as described above. We observed positive signals with antibodies against H3K9me2 and H3K27me2, without significant differences in ChIP-QPCR signals between young leaves and husk leaves (Figure 5d and data not shown). No signal was obtained in ChIP experiments with antibodies against H3K4me2, H3ac and H4ac (Figure 5d and data not shown). These data show that the copia sequence can be used as a positive ChIP control for histone modifications correlated with repressed chromatin, and a negative control for histone modifications correlated with active chromatin.

(5) Data normalization

The ChIP and QPCR procedure consists of many steps that can influence the final results. Before the obtained data can be interpreted, the variation caused by all these steps must be taken into account. The QPCR data have to be normalized for differences in the amount of input chromatin, precipitation efficiency and variation in the recovery of DNA after the ChIP. Importantly, normalization only serves to correct for this technical variation, it should not affect biological variation. The QPCR data can be normalized by various methods. Since normalization precedes data interpretation, the chosen normalization method can considerably influence the final conclusions. In a high quality ChIP experiment, with an optimal signal-to-noise ratio, the normalization method will not affect the biological interpretation, but in practice, experiments are often not optimal. This makes the choice for the appropriate normalization method essential for correct data interpretation.

The normalization step is often an underestimated part of ChIP experiments. In the literature there is no consensus on how to normalize ChIP-QPCR data, and as a result numerous different normalization methods are being used. Although the perfect normalization method does not exist, some are clearly more prone to result in incorrect data interpretation than others.

The currently used normalization methods for ChIP analysis are background subtraction [ 45], percent of input (%IP [ 28]), fold enrichment [ 46], normalization relative to a control sequence [ 47] and normalization relative to nucleosome density [ 48]. The various methods will be discussed here, along with their most important advantages and disadvantages (Table 2). If the data needed for multiple normalization methods are available, it is possible to assess the influence of the various methods on the interpretation of the data. This enables choosing the most applicable method.

Table 2 ChIP-QPCR normalization strategies

No normalization

With this method, the absolute signals derived from ChIP-QPCR are not normalized. When using this method, it is assumed that the signals between different samples can be compared without normalization. This is only possible when the amount of input chromatin is very carefully quantified, the quality of the isolated chromatin is highly reproducible and washing and purification steps are performed with absolute accuracy. The advantage of this method is that it requires a minimum of PCR reactions per experiment. The disadvantage lies in the fact that, to be able to compare the results obtained with different samples, equal amounts of chromatin should be used in the ChIP procedure.

Background subtraction

With this method the background signals, as measured with the NoAb control, are subtracted from the signals obtained from the ChIP samples. It is assumed that by subtraction of the background signals only the true enrichment is shown. The major shortcoming of this method is the fact that the levels of background signal in the NoAb control samples may be different from those in the ChIP samples. These potential differences in background signal levels can be caused by variation in serum components and by differences in sample handling between NoAb controls and ChIP samples. The level of background QPCR signal can in addition differ between different chromatin samples, primer pairs or separate ChIP experiments. Especially if the signal obtained with particular antibodies is only moderately higher than the background, subtraction will influence data interpretation and should therefore not be done. When the background is very low, subtraction does not influence data interpretation. Nevertheless, the NoAb sample is an essential control for the interpretation of ChIP signals, as it indicates the background signal level for the various primers sets in the different samples. We therefore propose to show the ChIP signals, without background subtraction, side by side with the background signals in all cases.

Fold enrichment

This normalization method is also called 'signal over background' or 'relative to the no-antibody control'. With this method, the ChIP signals are divided by the NoAb signals, representing the ChIP signal as the fold increase in signal relative to the background signal. The assumption of this method is that the level of background signal is reproducible between different primer sets, samples and replicate experiments. We strongly recommend not to use this method, because the background signal levels do vary between primer sets, samples and experiments (discussed above). The main disadvantage of this method can best be illustrated with data from a hypothetical ChIP experiment. A ChIP sample that is analyzed with two different primer sets results in a signal of 1 for both primer sets. The background signal (as measured in the NoAb control) is 0.01 for one primer set, and 0.001 for the other. These are realistic values since in our experiments we regularly observe a difference in NoAb control signal level of 10 fold or more between different primer sets. When the 'fold enrichment' normalization method is used for this data set, the normalized signal for the first primer set would be 1/0.01 = 100 and for the second 1/0.001 = 1000. The normalized data now suggest a 10 fold difference in signal level between the two different sequences, solely as a result of the NoAb signal levels. In reality there may be no difference at all. Clearly, the use of this normalization method can result in a random over- or under-representation of the ChIP data.

% of input

In this method, the QPCR signals derived from the ChIP samples are divided by the QPCR signals derived from the input sample taken early during the ChIP procedure (Figure 1). It is assumed that the obtained ChIP and NoAb signal levels are directly related to the amount of input chromatin. The main disadvantage of this method is caused by differences in handling between the input and ChIP samples; the input sample is taken from the chromatin solution very early in the ChIP procedure and processed separately from the ChIP samples. Another drawback is that, when comparing different samples, this method does not correct for differences in chromatin contaminations that inhibit antibody-antigen interactions. When the %IP method is used, one should take care that the normalization only corrects for technical variation and does not mask biologically important variation.

Relative to control sequences

With this normalization method, the QPCR signal derived from the sequence of interest is divided by the signal derived from the positive sequence control(s), assuming that the chromatin structure at the control sequence does not differ between samples, and that consequently all signal variation is of technical origin. The advantage of this method is that the same ChIP samples are used to analyze sequences of interest and control sequences, eliminating variation caused by sample handling. The disadvantage of this method is that the development and characterization of positive control sequences for all histone modifications of interest can be laborious. This method requires consistent positive QPCR signals for the positive control sequence with all antibodies used. When using probe-based QPCR chemistries, multicolour multiplex QPCR can be used to measure the region of interest and the control sequence in the same tube. Importantly, when control sequences are used to correct for differences in immunoprecipitation efficiency between various tissues, thorough testing of the control sequences is required to determine if all tissues yield identical results.

Relative to nucleosome density

The ChIP signals can also be normalized relative to nucleosome density. In that case, the ChIP-QPCR signals obtained with a specific antibody are divided by the signal obtained with an antibody against an invariant domain of a histone, for example histone H3. This normalization method corrects for differences in ChIP signals that are caused by differences in the density of nucleosomes, rather than by changes in histone modification levels.

Histone modifications can only be detected at a specific DNA sequence region if this region is also wrapped into nucleosomes. For example, a relatively low H3K4me2 signal at the transcription start site of a gene, when compared to the flanking regions, can be due to a low nucleosome density rather than a decreased presence of the modification as such [ 31].

A major disadvantage of this method is that it is very complicated to quantitatively compare ChIP-QPCR signal levels obtained by two different antisera. As discussed in the Choosing antibodies section, every antibody preparation has different epitope binding kinetics that are only linear in a specific range of input chromatin. In addition, the relation between epitope concentration and resulting ChIP-QPCR signal may be different for different antibody preparations. For example, a two-fold change in H3core ChIP-QPCR signal may reflect a different change in epitope availability than a two-fold change in a given histone modification ChIP-QPCR signal. This makes normalization to nucleosome density error prone, as the normalized signal is affected in an unpredictable manner. When this method is used to compare chromatin samples from different sources (e.g. root chromatin with leaf chromatin), the normalized data may in addition reflect differences in chromatin quality instead of nucleosome density. As discussed in the Fold enrichment section, at sequence regions that do not yield a clear signal above background in an H3core ChIP-QPCR experiment, normalization relative to nucleosome density will affect the signal levels in unpredictable ways and should therefore not be used. It is important to realize that an H3core ChIP-QPCR experiment can also provide biologically significant data on its own, and should be judged as such before deciding to use it for data normalization.

Before choosing a normalization method, the advantages and drawbacks of each method should be considered. Additionally, the choice depends on the experimental setup and the quality and availability of specific controls. As best practice, we propose to normalize ChIP QPCR data by '%IP' or 'relative to control sequences'. In our hands, both methods can be reliably used with high quality measurements of at least four replicates of a ChIP-QPCR experiment. We propose that ChIP experiments are repeated several times and that the results are presented together with the background signal and standard error. We also propose that ChIP data obtained with antibodies to invariant histone domains, when available, are presented as separate data sets.

# data[].attributes.attachment_link.title
Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization - Plant Methods

# data[].attributes.attachment_link.description
Background Chromatin remodeling, histone modifications and other chromatin-related processes play a crucial role in gene regulation. A very useful technique to study these processes is chromatin immunoprecipitation (ChIP). ChIP is widely used for a few model systems, including Arabidopsis, but establishment of the technique for other organisms is still remarkably challenging.

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Mohn F., Weber M., Schübeler D., Roloff TC. (2009) Methylated DNA Immunoprecipitation (MeDIP). In: Tost J. (eds) DNA Methylation. Methods in Molecular Biology, vol 507. Humana Press. https://doi.org/10.1007/978-1-59745-522-0_5

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Part of the Methods in Molecular Biology book series (MIMB, volume 507)

Abstract

Methylated DNA immunoprecipitation (MeDIP) is a versatile immunocapturing approach for unbiased detection of methylated DNA. In brief, genomic DNA is randomly sheared by sonication and immunoprecipitated with a monoclonal antibody that specifically recognizes 5-methylcytidine. The resulting enrichment of methylated DNA in the immunoprecipitated fraction can be determined by PCR to assess the methylation state of individual regions. Alternatively, MeDIP can be combined with large-scale analysis using microarrays as a genome-wide experimental readout. This protocol has been applied to generate comprehensive DNA methylation profiles on a genome-wide scale in mammals and plants, and further to identify abnormally methylated genes in cancer cells.

Keywords

MeDIP methylated DNA DNA methylation genome-wide analysis epigenomics 5mC antibody CpG immunoprecipitation

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# data[].attributes.attachment_link.description
Part of the Methods in Molecular Biology book series (MIMB, volume 507) Methylated DNA immunoprecipitation (MeDIP) is a versatile immunocapturing approach for unbiased detection of methylated DNA. In brief, genomic DNA is randomly sheared by sonication and immunoprecipitated with a monoclonal antibody that specifically recognizes 5-methylcytidine.

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Use of Chromatin Immunoprecipitation To Clone Novel E2F Target Promoters | Molecular and Cellular Biology

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Use of Chromatin Immunoprecipitation To Clone Novel E2F Target Promoters | Molecular and Cellular Biology

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Weinmann, Amy S., Stephanie M. Bartley, Theresa Zhang, Michael Q. Zhang, and Peggy J. Farnham. 2001. "Use Of Chromatin Immunoprecipitation To Clone Novel E2F Target Promoters". Molecular And Cellular Biology 21 (20): 6820-6832. doi:10.1128/mcb.21.20.6820-6832.2001.

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Volume 21 , Number 20
15 October 2001

MATERIALS AND METHODS

Cell culture and promoter and mRNA analyses.

HeLa cells were grown in 50% alpha minimal essential medium and 50% Joklik's medium with 5% supplemental calf serum (HyClone) and 1% penicillin-streptomycin (Gibco). The cells were grown to a density of 2 × 10 ⁵ to 5 × 10 ⁵ per ml before being harvested for cross-linking experiments. For all chromatin immunoprecipitation experiments presented in this study, asynchronously growing HeLa cells were used. For analysis of transcriptional properties of the ChET (for chromatin-precipitated E2F target) 8 promoter, a fragment was obtained by PCR using primers having sequences complementary to the −477 to + 94 region of the ChET 8 promoter plus restriction sites. The ChET 9 promoter studies were performed with a 293-bp segment of the ChET 9 clone. The PCR fragment was digested and inserted in either orientation into the HindIII site in pGL2 basic (Promega). For analysis of promoter activity and responsiveness to E2F1, NIH 3T3 cells (American Type Culture Collection) were maintained and transfected as described previously ( 14). pCMVE2F1 and the control vector pcDNA3 were described previously ( 25). U937 cells were maintained and RNA was prepared as described previously ( 8). Human tissue was procured at the University of Wisconsin Surgical Pathology Department; as required by our institutional review board protocol, the identities of the patients were unknown. The excess tissue was frozen after surgery, stored at −70°C, and prepared as described previously ( 11). Reverse transcription (RT)-PCR analyses were performed as described previously ( 11). Details of the primers used and the required hybridization temperatures can be found on our website at http://mcardle.oncology.wisc.edu/farnham. All primers were synthesized at the University of Wisconsin Biotechnology Center.

Chromatin immunoprecipitation.

Formaldehyde (Fisher Scientific) was added at a final concentration of 1% directly to cell culture media of nonadherent log-phase HeLa cells. Fixation proceeded at 22°C for 10 min and was stopped by the addition of glycine to a final concentration of 0.125 M. The HeLa cells were collected by centrifugation and rinsed in cold phosphate-buffered saline. The cell pellets were resuspended in swelling buffer (10 mM potassium acetate, 15 mM magnesium acetate, 0.1 M Tris [pH 7.6], 0.5 mM phenylmethylsulfonyl fluoride, and 100 ng of leupeptin and aprotinin/ml), incubated on ice for 20 min, and then Dounce homogenized. The nuclei were collected by microcentrifugation and then resuspended in sonication buffer (1% sodium dodecyl sulfate, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1], 0.5 mM phenylmethylsulfonyl fluoride, and 100 ng of leupeptin and aprotinin/ml) and incubated on ice for 10 min. Prior to sonication, 0.1 g of glass beads (212- to 300-μm diameter; Sigma) was added to each sample. The samples were sonicated on ice with an Ultrasonics sonicator at setting 10 for six 20-s pulses to an average length of approximately 1,000 bp and then microcentrifuged. The chromatin solution was precleared with the addition of Staphylococcus aureus protein A-positive cells for 15 min at 4°C. Prior to use, the Staph A cells were blocked with 1 μg of sheared herring sperm DNA/μl and 1 μg of bovine serum albumin/μl for at least 4 h at 4°C. Precleared chromatin from 10 ⁷cells was incubated with 1 μg of affinity-purified rabbit polyclonal antibody or no antibody and rotated at 4°C for approximately 12 to 16 h. Antibodies used included E2F1 no. 05-379 (UBI), E2F2 C-20 no. SC-633X (Santa Cruz), E2F3 C-18 no. SC-878X (Santa Cruz), E2F4 C-20 no. SC-866X (Santa Cruz), E2F5 E-19 no. SC999X (Santa Cruz), E2F6 E-20 no. SC-8366 (Santa Cruz), RNA polymerase II (a gift from David Bentley), p107 C-18 no. SC318X, p130 C-20 no. SC 317X, and Rb C-15 no. SC-50X. Immunoprecipitation, washing, and elution of immune complexes was carried out as previously described ( 3). Prior to the first wash, 20% of the supernatant from the reaction with no primary antibody for each time point was saved as total input chromatin and was processed with the eluted immunoprecipitates beginning at the cross-link reversal step. Cross-links were reversed by the addition of NaCl to a final concentration of 200 mM, and RNA was removed by the addition of 10 μg of RNase A per sample followed by incubation at 65°C for 4 to 5 h. The samples were then precipitated at −20°C overnight by the addition of 2.5 volumes of ethanol and then pelleted by microcentrifugation. The samples were resuspended in 100 μl of Tris-EDTA (pH 7.5), 25 μl of 5× proteinase K buffer (1.25% sodium dodecyl sulfate, 50 mM Tris [pH 7.5], and 25 mM EDTA), and 1.5 μl of proteinase K (Boehringer Mannheim) and incubated at 45°C for 2 h. Samples were extracted with phenol-chloroform-isoamyl alcohol (25:24:1) followed by extraction with chloroform-isoamyl alcohol and then precipitated with 1/10 volume of 3 M NaOAc (pH 5.3), 5 μg of glycogen, and 2.5 volumes of ethanol. The pellets were collected by microcentrifugation, resuspended in 30 μl of H ₂O, and analyzed by PCR. A detailed protocol can be found at http://mcardle.oncology.wisc.edu/farnham.

PCR mixtures contained 2 μl of immunoprecipitate or 2 μl of a 1:100 dilution of the total sample; 50 ng of each primer; 0.88 mM MgCl ₂; 0.2 mM (each) dATP, dCTP, dGTP, and dTTP; 1× thermophilic buffer (Promega); and 1.25 U of Taq DNA polymerase (Promega) in a total volume of 20 μl. Following 32 to 35 cycles of amplification, the PCR products were run on a 1.0% agarose gel and analyzed by ethidium bromide staining. The PCR primers used to analyze target genes can be found on our web site ( http://mcardle.oncology.wisc.edu/farnham).

Cloning novel E2F targets.

Several modifications of the chromatin immunoprecipitation protocol were required for the cloning of novel target genes. The first modification occurred at the elution step. Instead of eluting twice using 150 μl of elution buffer each time, the immunoprecipitated chromatin was eluted from the Staph A cells once, using 30 μl of elution buffer. The eluate was then diluted with 270 μl of immunoprecipitation dilution buffer to a total volume of 300 μl, and a new aliquot of the same antibody as that used in the first immunoprecipitation was added for an overnight incubation at 4°C. The next morning, the samples were processed in the standard manner (i.e., they were washed and eluted, cross-links were reversed, and the samples were proteinase K digested, followed by phenol extraction and ethanol precipitation). At this point, an aliquot of the immunoprecipitated samples was used in a reaction with b- myb or dhfr primers to demonstrate that E2F targets had been selected. The remaining immunoprecipitated DNA was then treated with T4 DNA polymerase and cloned into either the zero-blunt vector (Invitrogen) or HincII-digested puc 19. Colonies having inserts were identified by restriction enzyme digestion using enzymes in the polylinker. Plasmids having inserts greater than 500 bp were chosen for further analysis. The sequence of each of the cloned fragments, details concerning the sequences of the primers used to analyze each clone, the required hybridization temperatures, and the product sizes can be found on our web site ( http://mcardle.oncology.wisc.edu/farnham). All primers were obtained at the University of Wisconsin Biotechnology Center.

Electromobility shift assays.

In vitro E2F DNA binding activity was assayed by incubating about 6 μg of HeLa nuclear extract with 2.5 μg of sonicated salmon sperm DNA and 2 μl of 5X-500 buffer (100 mM HEPES [pH 7.4], 500 mM KCl, 5 mM MgCl ₂, 0.5 mM EDTA, 35% glycerol, and 5 mM NaF) in a total volume of 18 μl for 10 min at room temperature. Either a 34-bp double-stranded oligonucleotide containing the E2F site from the b- myb promoter or a 22-bp double-stranded oligonucleotide containing the E2F site from ChET 9 (both of which were end labeled using T4 DNA kinase and [γ- ³²P]ATP as described previously [ 28]) was then added in 2 μl of water, and the incubation continued for 20 min. The reactions were electrophoresed for 2 h on a 4% polyacrylamide gel that had been preelectrophoresed for 30 min. When competition assays were performed, the competitor DNA was included in the first incubation at a 50-fold molar excess to the labeled probe.

RESULTS

In vivo isolation of E2F binding sites.

Conventional target gene identification methods, such as microarray analysis or subtractive hybridization, examine changes in gene expression profiles. Although valuable information can be obtained from such studies, the disadvantage of using these techniques to clone target genes of transcription factors is that the observed changes may in fact be the result of indirect pathways influencing gene expression patterns. For example, approximately 7% of the mRNAs on a human cDNA microarray responded to overexpression of an E2F ( 31). However, promoter analyses were not performed on these genes. Therefore it is not known if the genes in that study are regulated by promoters that contain consensus E2F binding sites or if the promoters are directly bound by E2F. In contrast, one major advantage of utilizing the chromatin immunoprecipitation method to identify novel target genes is that it selects for sites bound by the transcription factor of interest, thus eliminating the problem of indirect effects. Therefore, we have chosen to use chromatin immunoprecipitation as an unbiased approach to identify in vivo E2F binding sites (Fig. 1 A).

Fig. 1. (A) Schematic of the E2F chromatin immunoprecipitation cloning procedure. (B) Graphical representation of the results of a chromatin immunoprecipitation experiment measuring E2F binding at the Myc promoter, Myc exon 2, and Hox3D exon 2. Scores representing homology to the E2F consensus site as determined by computer analysis (21) are shown at the top of each graph. They axis represents Imagequant quantitation of the amount of specific PCR products expressed as the percentage of antibody binding versus the amount of PCR product obtained using a standarized aliquot of input chromatin. The signal in the no-antibody lane was subtracted from each sample as a nonspecific binding background. The E2F family members used in the immunoprecipitation are shown on thex axis.

Briefly, following formaldehyde cross-linking of cells, chromatin is isolated and sheared to a desired length by sonication. Immunoprecipitation proceeds with an antibody to a factor of interest to selectively precipitate that protein and any DNA fragment cross-linked to it. Thus, DNA sequence elements associated with the desired protein in the context of the cellular environment are enriched in the immunoprecipitated sample. After reversal of the formaldehyde cross-links and purification of the DNA, the precipitated DNA fragments are cloned into a vector for isolation and further characterization of factor binding sites and functional relevance. Although the protocol for cloning E2F targets is similar to the standard chromatin immunoprecipitation assay that has been previously described ( 3, 33, 49), several modifications were made to the assay. Importantly, we performed two sequential chromatin immunoprecipitations using the same antibody for both the first and second steps. In preliminary studies, we found that a large number of nonspecific fragments were cloned if only one immunoprecipitation was performed. Therefore, the second immunoprecipitation was used to decrease the amount of nonspecific DNA present to enable more efficient cloning of the specific fragments. Also, a portion of the immunoprecipitated samples was retained prior to cloning to analyze known target genes as a positive control for the immunoprecipitation. Other modifications important for cloning the immunoprecipitated products can be found on our web site ( http://mcardle.oncology.wisc.edu/farnham).

Previous studies have demonstrated the validity of using the chromatin immunoprecipitation protocol to identify site-specific interactions of transcription factors and promoter DNA in the context of a living cell. Importantly, we have shown that binding of E2F to target promoters is site specific. For example, the chromatin immunoprecipitation assay has been used to demonstrate that E2F4 binds with high affinity to the dhfr promoter (which is known to be regulated by E2F) but does not bind to the cad promoter (which is a Myc target gene) ( 3). Also, in a previous report ( 49) we showed that a site-specific mutation in the dhfr promoter eliminates binding of E2F4, as monitored by the chromatin immunoprecipitation assay. In addition, high-affinity binding of E2F family members to the myc promoter is abolished when a point mutation is introduced into the E2F site of that promoter ( 2). Finally, we have also shown that E2Fs do not bind to promoters which are regulated by liver-specific transcription factors (C. R. Graveel and P. J. Farnham, unpublished data). Clearly, the chromatin immunoprecipitation assay has been useful in demonstrating that not all cellular promoters bind E2Fs and that those that do require a specific site on the DNA for high-affinity in vivo binding. However, we felt that additional controls were needed prior to using this assay to clone novel targets. Namely, we wished to clone regulatory E2F binding sites and not clone random, nonfunctional E2F binding sites present in nonpromoter regions of the genome. Previous analysis of promoter and exon 2 databases suggested that E2F sites are found at a much higher frequency in promoters ( 21). However, sequences having high-score matches to the E2F consensus site (scores of 0.86 or better in the computer analysis) can be found in nonpromoter regions as well. To explore whether E2F is bound to these sites in living cells, we identified two such consensus sites located in the second exon of the myc and the hox3Dgenes. Because the myc promoter has previously been shown to be an E2F target ( 30, 34, 50), we examined binding of E2F family members to the exon sites in comparison to binding at the E2F site in the Myc promoter. As shown in Fig. 1B, E2F binding to the myc gene promoter is at much higher levels than binding to the myc and hox3D exon 2 regions in the same chromatin sample, even though the score match to the consensus E2F site is very high in the exon 2 regions.

Having assured ourselves that the chromatin immunoprecipitation assay can be used to detect promoter-specific and site-specific binding of E2F, we performed immunoprecipitations from HeLa cell chromatin using antibodies against either E2F1 or E2F4 and proceeded with the cloning procedure. We chose clones having inserts of 500 bp or greater and examined 11 clones obtained by immunoprecipitation with the E2F1 antibody and 7 clones obtained by immunoprecipitation with the E2F4 antibody for further analysis. The first step in the characterization of the ChET clones was to determine which ones contained bona fide E2F binding sites and which were false positives.

Confirmation of E2F binding.

In vitro assays may bias results towards identifying sites which closely resemble the known consensus E2F binding site. In addition, any large genomic fragment may, by chance, contain a sequence resembling an E2F site and be scored as a positive in an in vitro assay. Therefore, we began confirmation of our cloned fragments using an in vivo assay. The ChET clone inserts were sequenced, and primers were made for use in chromatin immunoprecipitation experiments. Because the E2F binding site could be anywhere within the cloned fragment, primers were designed to analyze both ends of each clone. HeLa chromatin was immunoprecipitated using antibodies to E2Fs 1 to 6; a no-antibody negative control was also performed (Fig. 2). As a positive control, binding of E2F family members to the dhfr promoter was examined. For each primer set, a standardized aliquot of the input chromatin (total) was also analyzed. The ratio of the signals detected in the lanes containing samples immunoprecipitated by the various antibodies to the signal detected in the total lane allows a relative comparison representing the degrees of occupancy at the different sites. Figure 2 shows the results of a representative experiment using the primer sets that showed the highest-affinity E2F binding for each clone.

Fig. 2. Confirmation of ChET clones by examining E2F binding in vivo. A representative chromatin immunoprecipitation experiment with HeLa cells is shown. Immunoprecipitation proceeded utilizing antibodies (Ab) against E2F1 (lane 1), E2F2 (lane 2), E2F3 (lane 3), E2F4 (lane 4), E2F5 (lane 5), and E2F6 (lane 6) or no antibody (lane 8). Following DNA purification, samples were subjected to PCR with primers designed for the individual E2F clones or the dhfr promoter as a control (labeled on the right). A portion of the total input was also examined by PCR (lane 7).

We began by testing the clones obtained using an E2F4 antibody in the immunoprecipitation step. Seven clones from the E2F4 immunoprecipitation were analyzed for E2F binding in vivo (Fig. 2). Of these, one clone (ChET 2) was found to be present in samples in which no antibody was added to the immunoprecipitation reaction mixture, indicating that binding was nonspecific. The remaining six clones were all bound specifically by E2F family members in numerous independent chromatin immunoprecipitation experiments. Although several of these clones (e.g., ChET 1) were of modest affinity, other clones (e.g., ChET 4) showed robust binding of E2Fs. Eleven clones obtained by immunoprecipitation with an E2F1 antibody were also examined to determine whether they were bound by E2Fs in vivo. We were unable to optimize primer sets to four of these clones for analysis in subsequent chromatin immunoprecipitation assays; these four clones will be included in future studies if optimal primer sets can be empirically determined. Therefore, we were left with seven clones for in vivo analysis. We could not confirm binding of E2Fs to four of these clones in subsequent chromatin immunoprecipitation assays; an example of one such false positive is shown below (see Fig. 4, ChET 10). However, three of the seven E2F1 clones contained bona fide E2F binding sites as determined by chromatin immunoprecipitation analysis. The ChET 8 and ChET 9 clones both contained high-affinity E2F binding sites in comparison to the binding detected at the well-characterized dhfr promoter (Fig. 2). Binding to the third E2F1 clone was of modest affinity (data not shown). In summary, of the 14 clones that we were able to examine using in vivo assays (7 obtained using an E2F1 antibody and 7 obtained using an E2F4 antibody), 9 were confirmed to contain bona fide in vivo E2F binding sites (Table 1). Although false positives are unavoidable, we believe that the two sequential immunoprecipitation reactions greatly enriched for clones containing bona fide E2F binding sites. It is important to note that the in vivo binding of E2Fs to the ChET clones has been confirmed in numerous experiments. For example, the experiments shown in Fig. 2 and below (see Fig. 4) are completely independent from each other and from the chromatin immunoprecipitation experiment used to clone the fragments. We have also confirmed binding of E2Fs to ChET 4, ChET 8, and ChET 9 in other human cell types (data not shown).

Table 1. Summary of ChET cloning

Parameter

Value

E2F4a

E2F1a

Total (%)

No. of fragments analyzed

7

7

14

No. of false positives

1

4

5 (36)

No. of clones specifically bound by E2Fs

6

3

9 (64)

Antibody to the indicated E2F was used to clone the fragments.

Either an E2F1 or an E2F4 antibody was initially used for the cloning procedure; however, we found that the cloned sites did not reveal an E2F binding pattern to suggest family member binding specificity. Rather, binding of multiple E2F family members to each clone was detected in vivo (Fig. 2). These results, suggesting a lack of DNA binding specificity among the different E2Fs, are similar to those of our previous studies of known E2F target genes ( 49). E2F family members contain a highly conserved DNA binding domain; therefore, it is not surprising that these family members have the ability to bind to the same sequence in vivo. It is unlikely that our results indicate that multiple E2F family members are simultaneously binding to the same site, but the more likely explanation is that a dynamic exchange occurs at a given site and various E2Fs are trapped in different cells during the cross-linking procedure. There are precedents for this hypothesis, as dynamic exchange of the glucocorticoid receptor has been demonstrated in living cells ( 29). With this in mind, it is worth noting that the cloned sites can be separated into three basic categories. The first type (e.g., ChET 9) showed an in vivo binding pattern very similar to that of the dhfr promoter, i.e., strong binding of E2F1 to E2F4 and very little binding of E2F5 or E2F6. The second type (e.g., ChET 4 and ChET 8) showed strong binding of E2F1 to E2F4 and little binding of E2F5 but detectable binding of E2F6. Finally, the third type of site (e.g., ChET 1) showed equal low-affinity binding of E2F1 to E2F6. The significance of these distinct binding patterns is unknown, and further analysis will be required to elucidate the functional consequences, if any.

Genomic organization of ChET clones.

We next analyzed the sequences of the cloned fragments to determine if anything was known about their identities. Three of the high-affinity clones (ChET 4, ChET 8, and ChET 9) contained extremely GC-rich sequences, which is a hallmark of many E2F-regulated promoters. In addition, the PromoterInspector program ( 39) detected a promoter region in ChET 9 and the CpG Promoter program ( 15) detected a promoter-associated CpG island in ChET 4 and ChET 8. Most E2F-regulated promoters have E2F binding sites located in close proximity to the start site of transcription ( 21). If our cloned fragments do, in fact, represent promoters, then it is possible that the sequences just downstream of the E2F sites correspond to transcribed regions. To test this hypothesis, we first identified the region of the human genome corresponding to the cloned fragments and then compared several kilobases of sequence on either side of the cloned fragments to the GenBank database for a potential cDNA match (Fig. 3). Interestingly, we found that a previously identified but uncharacterized mRNA begins within the ChET 8 clone approximately 130 bp from one end. Also, a previously uncharacterized mRNA begins within ChET 9 (although the exact 5′ end of this mRNA has not yet been determined). Finally, the 5′ end of the mRNA encoding Beclin 1, a candidate tumor suppressor protein, is contained within the ChET 4 clone.

Fig. 3. Genomic organization of the ChET clones. GenBank database searches were performed with the sequences corresponding to ChET 4, ChET 8, and ChET 9. After the locations of the clones were determined, further Blast searches were performed examining the sequences immediately adjacent to the cloned fragments. The locations of adjacent mRNAs are indicated by bent arrows. Positions of consensus Sp1 (rectangles) and E2F (oval) sites are also shown.

In summary, it appears that the three high-affinity E2F binding sites all correspond to promoter regions: ChET 8 is the promoter region for a 6,049-nt mRNA termed KIAA0254 (accession number D87443), which encodes a 1,009-amino-acid protein with no homology to other known proteins; ChET 9 is the promoter region for a 4,441-nt mRNA termed KIAA0160 (accession number D63881), which encodes an 803-amino-acid protein having extensive homology to the Drosophila protein Suppressor of zeste 12 [Su(z)12]; and ChET 4 is the promoter for a 2,098-nt mRNA encoding Beclin 1 (accession number AF139131), which is a candidate tumor suppressor gene. Of the five remaining E2F4 ChET clones, four can be identified in the human raw-sequence database. We have searched the GenBank database using several kilobases of surrounding sequence but have not identified mRNAs associated with these clones. Therefore, it is still unclear whether the remaining E2F4 ChET clones correspond to promoter regions.

Characterization of the novel E2F binding sites.

The next step in the characterization of the ChET clones was to determine the compositions of the protein complexes recruited to the clones in vivo. E2Fs bind directly to Rb, p107, and p130, and we have previously shown that these proteins are components of the transcription complexes formed on E2F target promoters in vivo ( 49). E2Fs also bind to basal transcription factors, such as TBP and TFIIH ( 35); therefore, it is likely that components of the RNA polymerase II transcription complex are also recruited to E2F target promoters. To examine these possibilities, chromatin immunoprecipitation experiments were performed using antibodies to E2Fs, pocket proteins, and RNA polymerase II (Fig. 4). For these and the remaining experiments, we chose to focus on the three clones which have the highest levels of in vivo E2F binding, ChET 4, ChET 8, and ChET 9. The data from the chromatin immunoprecipitation experiment shown in Fig. 4suggest that, similar to known E2F binding sites, such as dhfr, the novel E2F binding sites also recruit pocket proteins. Interestingly, the binding profiles for the pocket proteins observed at the three novel clones vary in comparison to that observed at the dhfr promoter. The dhfr promoter is bound mainly by p107 and p130, with very little bound Rb, whereas the novel clones recruited almost equivalent levels of the three pocket proteins. Because the use of HeLa cells may have given altered pocket protein binding due to the expression of the viral E7 protein, we also examined pocket protein binding to the dhfr promoter and to the three ChET clones in U937 cells. We found very similar recruitment of pocket proteins to the E2F target promoters in the U937 cells and in HeLa cells (data not shown). In addition, the novel E2F binding sites recruited RNA polymerase II, suggesting that the three high-affinity clones indeed represent promoter sequences.

Fig. 4. Characterization of the protein complexes bound in vivo to the ChET clones. A chromatin immunoprecipitation experiment was performed in HeLa cells utilizing antibodies (Ab) to E2F1 (lane 1), E2F4 (lane 2), p107 (lane 3), p130 (lane 4), Rb (lane 5), RNA polymerase II (Pol II; lane 6), or no antibody as a control (lane 8). An aliquot of the total input is also shown (lane 7). Primers to the ChET clones or the DHFR promoter were used in PCRs for analysis.

We wished to further characterize a subset of the ChET promoter clones. We chose the ChET 8 and ChET 9 clones for an initial promoter analysis. If we are correct in assuming that the ChET 8 clone represents the promoter for the KIAA0254 mRNA, then it should have promoter activity when inserted in the correct orientation with the transcription start site upstream of the luciferase cDNA. The ChET 8 fragment was cloned in both orientations (forward and reverse) upstream of the luciferase cDNA and transfected into NIH 3T3 cells, and promoter activity was measured. The cdc2 promoter-luciferase reporter construct was used in these experiments as a positive control. As shown in Fig. 5A, only the forward orientation of the ChET 8 fragment showed high promoter activity, whereas the reverse orientation did not. In fact, the ChET 8 promoter was considerably more active than the cdc2 promoter, which is a strong E2F-regulated promoter. Importantly, the orientation of the ChET 8 fragment that showed promoter activity was the correct orientation to drive KIAA0254 mRNA transcription.

Fig. 5. Transient-transfection analysis of ChET promoter-luciferase reporters. (A) A transient-transfection experiment in NIH 3T3 cells was performed with a segment of ChET 8 cloned in either the forward or reverse orientation upstream of luciferase. They axis of the graph represents the relative luciferase units, with the transfected material shown on the xaxis. pGL2 represents the luciferase vector lacking a promoter. (B) A transient-transfection analysis was performed with the ChET 8 promoter-luciferase reporter transfected into NIH 3T3 cells in the presence of 2 μg of an E2F1 expression vector (cytomegalovirus [CMV] E2F1) or the pCDNA3 vector as a control. Thecdc2 promoter-luciferase reporter construct was used as a control in both panels A and B. (C) Transient-transfection analysis of the ChET 9-luciferase reporter construct was performed in NIH 3T3 cells. A graphical representation of the results is shown with thedhfr promoter used as a positive control. (D) Overexpression of E2F1 upregulates ChET 9 promoter activity. Cotransfection experiments were performed containing 2 μg of a CMV E2F1 expression construct with the ChET 9-luciferase construct or thedhfr-luciferase reporter vector as a control. Results of the luciferase assay are shown in the graph as indicated for panel A.

We have previously shown that E2F site-containing promoters can be activated by cotransfection with E2F1 ( 25). To determine if binding of E2F influences the transcriptional activity of the ChET 8 promoter, we cotransfected the ChET 8 promoter-luciferase reporter construct with an E2F1 expression construct, again using cdc2-luciferase as a positive control (Fig. 5B). We found that, as expected, E2F1 overexpression activated the cdc2 promoter. In contrast, E2F1 repressed transcription from the ChET 8 promoter. Although the exact mechanism by which E2F1 represses the ChET 8 promoter is still unknown, preliminary analyses indicate that squelching (i.e., the sequestration of coactivators and/or general transcription factors by a transactivation domain) is most likely not the mechanism. For example, an E2F1 construct with the entire transactivation domain deleted is still a potent repressor of ChET 8 promoter activity (data not shown). The potential relevance of these findings will be discussed further below (see Discussion).

The ChET 9 fragment was also cloned into the luciferase reporter vector for further analysis to determine if indeed this cloned sequence contains promoter activity. A transient transfection analysis of the ChET 9 luciferase reporter construct demonstrated that the ChET 9 clone has promoter activity (Fig. 5C) which is approximately equal to the activity of the dhfr promoter (Fig. 5D). In addition, the overexpression of E2F1 stimulates ChET 9 promoter activity (Fig. 5 D). An extensive characterization of the ChET 9 promoter is in progress; however, it is worth noting that deletion of the region containing the consensus E2F site eliminates the E2F1-mediated transactivation (data not shown). These results indicate that the ChET 9 clone has promoter activity and indeed contains an E2F site which is functional in a transient overexpression system.

Localization of potential E2F sites.

Visual inspection of the sequences corresponding to the three cloned promoter fragments bound by E2F family members in vivo revealed that only one clone, ChET 9, contains a consensus E2F site. Using a computer program which has previously been shown to effectively predict consensus E2F binding sites ( 21), we next examined the clones for matches to the E2F consensus sequence. The cutoff previously used for identifying E2F sites was a 0.86 similarity to the consensus ( 21). Using this criterion, once again the only clone containing a consensus E2F site was ChET 9. We thought that it might be possible to identify the E2F binding sites within the ChET clones by using an in vitro assay. Therefore, electromobility shift assays (EMSA) were performed using the well-characterized E2F binding site in the b- myb promoter as a probe. Two bands (complexes 1 and 2) which represent specific protein-DNA complexes were observed, as determined by antibody disruption (Fig. 6A) and competition with the unlabeled probe (Fig. 6B); an additional nonspecific band (complex 3) was also observed in most reactions. Because each of the six E2Fs heterodimerizes with DP-1, we used the DP-1 antibody to show that complex 2 contains an E2F-DP heterodimer (Fig. 6A). Although complex 1 is specifically competed by the probe (Fig. 6B), the DP-1 antibody did not disrupt it. We (reference 24 and data not shown) and others ( 41) have previously shown that a low-mobility complex which binds specifically to E2F probes cannot be identified by using antibodies to the different E2F family members. It is possible that this complex represents an uncharacterized E2F family member, or the epitope may be obscured by another protein. To distinguish these possibilities, we employed antibodies to the pocket proteins. However, antibodies to the pocket proteins did not disrupt complex 1 (Fig. 6 A). Therefore, the identities of the proteins composing complex 1 are still unknown.

Fig. 6. In vitro EMSA of cloned fragments. In each panel (arrows), complex 3 represents a nonspecific band, complex 2 indicates an E2F-DP complex, and complex 1 indicates a complex containing proteins that have not yet been identified. (A) Supershift EMSA was performed to determine the components of the gel-shifted complexes. Reaction mixtures containing HeLa nuclear extract were incubated with an antibody (Ab) to Sp1 (lane 2), DP-1 (lane 3), p130 (lane 4), p107 (lane 5), or Rb (lane 6) or no antibody (lane 1) followed by incubation with the b-myb E2F site labeled as a probe. (B) EMSA competition experiments using the E2F site from the b-myb promoter as a probe. Oligonucleotides corresponding to the unlabeled probe (lane 2), a fragment spanning the ChET 8 transcription start site (lane 3), an oligonucleotide corresponding to the consensus E2F site from ChET 9 (lane 4), or the ChET 9 fragment (lane 5) were used as competitors. (C) EMSA using the ChET 9 E2F site as a probe. A double-stranded oligonucleotide containing the consensus E2F site within the ChET 9 fragment (the sequence is shown in Fig. 3) was radiolabeled and used for EMSA. The probe was incubated with HeLa nuclear extract and the unlabeled probe (lane 3), a DP-1 antibody (lane 4), or extract alone (lane 2). Lane 1 represents an aliquot of the probe without extract incubation. An arrow to the right of the gel image indicates the specific E2F-DP complex.

There are no consensus E2F binding sites located in the ChET 8 clone; therefore, it was not apparent which region was responsible for the recruitment of E2F to the promoter region. Because E2F sites are often found within 50 bp of the transcription start site ( 21), we prepared a fragment which surrounds the transcription start site for use as a competitor in an EMSA competition experiment (Fig. 6B). Competition was observed by using this promoter-proximal fragment; other regions of the ChET 8 clone did not compete the probe (data not shown). Further analyses will have to be performed to determine the exact location of the E2F site within the ChET 8 clone. Because competition was not complete in the in vitro EMSA experiments, an alternative in vivo method for assaying binding activity may need to be developed to localize the E2F site within the cloned fragment. Although a complete mutational analysis of the promoter region is beyond the scope of this initial study, we have recently shown that the region required for responsiveness to E2F in the transient-expression assay (Fig. 5B) resides in the promoter-proximal fragment shown as a competitor in Fig. 6 B (unpublished data).

EMSA competition experiments utilizing the oligonucleotide corresponding to the consensus E2F site in the ChET 9 promoter revealed that this site is an efficient competitor of the DP-E2F-DNA complex formed on the b- myb E2F site (Fig. 6B, lane 4). Interestingly, additional sequences located in other regions of the ChET 9 fragment are required to compete binding of complex 1 (Fig. 6B, lane 5). To confirm that E2F has the ability to bind directly to the E2F site identified in ChET 9, we performed EMSA experiments utilizing a radiolabeled probe containing the E2F site located in ChET 9. As shown in Fig. 6C, a single lower-mobility complex is detected when nuclear extract is incubated with the ChET 9 probe. The addition of a DP-1 antibody to this reaction ablated the complex, indicating that this complex contains a DP-1-E2F heterodimer. Thus, the consensus site in ChET 9 competes binding to the E2F site in the b- myb promoter and binds to a protein complex which can be disrupted by a DP-1 antibody. Taken together with the promoter-reporter assays, our results suggest that the consensus E2F site in ChET 9 contributes to in vivo binding and E2F responsiveness.

Characterization of expression patterns.

Many E2F-regulated promoters display cell cycle stage-specific transcription patterns. However, almost without exception, the previously identified E2F-regulated promoters were identified from a pool of promoters already known to be cell cycle regulated ( 42). It seemed possible that promoters identified by using an unbiased approach might show different transcriptional regulation. Therefore, we monitored the expression levels of the mRNAs driven by ChET 4, ChET 8, and ChET 9 in a cell differentiation system. U937 cells were forced to differentiate by treating them with retinoic acid for 5 days, and then RNA was prepared from log-phase (growing) and arrested (differentiated) cells. As a control, we monitored the expression of E2F1, an E2F-regulated gene that has been shown to be downregulated when growing cells exit the cell cycle ( 14, 18, 32, 43). As expected, levels of E2F1 mRNA decreased upon differentiation and cell cycle arrest (Fig. 7A). In contrast, the levels of mRNA for the three novel clones did not decrease significantly in the U937 cell population upon differentiation, which suggests that these promoters are not cell cycle regulated. These data indicate that E2F family members may regulate both cell cycle- and non cell cycle-responsive promoters. To determine whether another unbiased approach would also yield constitutively active E2F-regulated promoters, we examined the mRNAs for two promoters that were predicted to be regulated by E2F from a computer-assisted identification of consensus E2F binding sites in the promoter regions. The large subunit of RNAPII and XRCC2 were both identified in a previous study which scanned the eukaryotic promoter databases for E2F target promoters ( 21). Chromatin immunoprecipitation analysis indicated that indeed the promoters for RNAPII large subunit and XRCC2 are bound by E2F (data not shown). Analysis of mRNA levels in the U937 cells indicated that one of the two computer-identified genes was constitutively expressed whereas the other was downregulated upon differentiation. Therefore, four of the five E2F-bound promoters identified by using unbiased approaches are not regulated upon differentiation of U937 cells. It is also important to note that although the amount of E2F1 declines during differentiation of U937 cells, the overall amount of E2F activity remains high due to the constitutive expression of E2F4. In fact, we have shown that the amount of E2F4 bound to the ChET promoters is unchanged after differentiation of U937 cells (data not shown). Perhaps only those E2F target genes which are uniquely responsive to E2F1 versus E2F4 will show a decline in activity upon differentiation.

Fig. 7. mRNA expression profiles of the three high-affinity ChET clones. (A) RT-PCR analysis of mRNA expression levels in RNA obtained from either U937 log-phase (lane 1) or differentiated (diff; lane 2) cells. RT-PCR primers complementary to E2F1, ChET 9, ChET 8, ChET 4, RNA polymerase (RNAP) II, or XRCC2 were used as indicated on the right. A water control is shown in lane 3. (B) RT-PCR analysis of RNA from either normal (N) colon (lane 1) or colon tumor (T; lane 2). Primer sets to the specific mRNAs are indicated. (C) RT-PCR analysis of ChET 9 mRNA expression in the RNA obtained from either human normal colon (lane 1), colon tumor (lane 2), normal liver (lane 3), or liver tumor (lane 4). The normal colon and colon tumor samples are the same as those shown in panel B with GAPDH primers added as a loading control.

E2F target genes have been suggested to be critical regulators of cell growth control. Therefore, we also examined whether the expression of ChET 4, ChET 8, and ChET 9 is altered during neoplastic transformation. We first compared the levels of mRNAs in samples of human normal colon and colon tumor taken from the same patient (Fig. 7B). In RT-PCR analysis, ChET 4 had slightly higher levels of mRNA expression in tumor tissue, ChET 8 showed a slight decrease in mRNA levels in the tumor sample, and ChET 9 was significantly upregulated in the colon tumor tissue. To determine if upregulation of ChET 9 mRNA was specific to colon tumors, we also compared normal liver and liver tumor mRNA levels. To control for equivalent RNA sample concentrations, primers for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were included in the reaction mixtures. The results show that ChET 9 mRNA was upregulated in both tumor types (Fig. 7 C), which suggests that ChET 9 expression may be generally deregulated in human tumors. We have also confirmed that ChET 9 mRNA is upregulated in eight of nine additional human colon tumor samples (unpublished data).

DISCUSSION

To our knowledge, this is the first demonstration that the chromatin immunoprecipitation assay can be used to clone promoters which are direct in vivo targets of a mammalian site-specific DNA binding protein. This provides a powerful new approach to examine direct transcription factor targets in an unbiased manner which does not rely on previous characterization of a consensus sequence or a prior knowledge of gene expression patterns. Although others have used similar approaches to isolate genomic fragments ( 6, 36, 38), those studies did not use subsequent experiments to confirm in vivo binding of the factor of interest to the isolated DNA. Due to this lack of in vivo confirmation, it is difficult to assess the validity of the previous protocols. Also, sequence analysis of the clones isolated in the previous studies indicated that the cloned fragments corresponded to nonpromoter regions, such as introns ( 5, 10, 22, 45, 46). One study did find that 3 out of 43 clones isolated after in vitro incubation of genomic DNA with purified Ets-1 protein were promoters; however, the authors did not confirm in vivo binding of ETS1 to these 3 clones or to the other 40 isolated clones. Therefore, it is difficult to be sure if any of the clones in that study were bona fide in vivo targets of ETS1.

Utilizing the chromatin immunoprecipitation assay to clone fragments bound by E2F family members, we found that 64% (9 of 14) of the clones characterized were bona fide in vivo E2F binding sites. Characterization of the three highest-affinity clones revealed that they correspond to promoter regions (Fig. 8), providing validation that novel E2F-regulated promoters can be isolated by using this protocol. Future studies will be performed to determine whether any of the remaining clones are promoters. A recent study using high-density microarray analysis ( 31) found that about 7% of the mRNAs represented on the microarray were responsive to overexpression of E2Fs. These results suggest that a high percentage of mammalian genes might be regulated by direct binding of E2F to the promoter region. If this estimate of the number of E2F target genes is correct, then it is not surprising that we did not isolate one of the several dozen well-characterized E2F target promoters in the set of nine positive clones that we analyzed. However, one of our ChET clones (ChET 9, which corresponds to the promoter region of the KIAA0160 gene) was shown to be upregulated by E2F overexpression in the microarray analysis ( 31). The fact that this gene was isolated by two independent screening methods for E2F target genes provides strong evidence that this promoter is indeed a direct target of E2F family members and that the chromatin immunoprecipitation cloning technique can identify E2F-regulated promoters.

Fig. 8. Summary of information obtained relating to the ChET promoter clones.

One of the E2F4 clones, ChET 4, displayed high-affinity E2F binding in vivo and corresponded to the promoter region for the beclin 1 gene. Beclin 1 was isolated through its ability to interact with bcl-2 and has been postulated to possess tumor suppressor activity in breast cancer ( 1). It is interesting that the gene for a potential tumor suppressor protein was isolated as an E2F target gene because E2F regulation is thought to play a significant role in tumorigenisis. Further experiments examining the nature of the role E2F plays in Beclin 1 regulation may provide further insight into the role of E2F in tumor development.

We found that two of the high-affinity E2F binding clones did not contain E2F sites which closely matched the consensus sequence. It is important to note that others have previously shown that site-specific DNA binding proteins can regulate transcription through sequence elements that diverge from the consensus. For example, CREB, Ets-1, and AML1 can regulate expression of the human T-cell receptor beta chain promoter through nonconsensus binding sites ( 12) and a nonconsensus site mediates regulation of the atrial natriuretic factor by serum response factor ( 13). Computer inspection suggests that the ChET clones may contain multiple low-affinity E2F sites, each of which diverges from the known consensus. Perhaps a combination of weak binding sites allows for cooperative recruitment of the E2F complex in vivo. It is also possible that the promoter context may greatly influence E2F binding efficiency within the cellular environment. We have previously shown that some (e.g., CCAAT and YY1) but not all (e.g., Oct1, Ap2, and NF1) transcription factor sites can synergize with E2F sites to activate transcription ( 47). It is possible that this synergy was mediated by cooperative DNA binding. Also, others have shown that Sp1 can physically interact with E2F family members and that binding of Sp1 can influence the occupancy of a nearby E2F site ( 20, 27). Each of the three characterized ChET clones contains at least one consensus Sp1 binding site (Fig. 3). Finally, others have shown that E2Fs can interact with other sequence-specific DNA binding proteins, such as C/EBPα ( 17, 44). Interestingly, we have recently shown that E2F1 can be recruited to promoters which contain C/EBPα binding sites but lack E2F consensus sites (Graveel and Farnham, unpublished). It remains to be determined if C/EBPα and/or other protein-protein interactions are mediating the recruitment of E2F to the promoters we have cloned. However, recruitment of E2F through the recognition sequence of another DNA binding protein could explain why some of the cloned fragments failed to show robust competition of a consensus E2F site in vitro.

To date, the majority of well-characterized E2F target promoters have been shown to be cell cycle regulated and activated by E2F overexpression. In contrast, our three novel E2F target promoters are constitutively expressed in growing versus differentiated U937 cells. It is perhaps not surprising that E2F target promoters isolated using an unbiased approach show expression profiles different from those of the well-characterized E2F target promoters. According to microarray analyses, hundreds of genes are regulated by E2F family members ( 16, 31). It is highly unlikely that this large number of mRNAs, which encode proteins having highly diverse biological functions, will all show exactly the same expression pattern in all cell types.

It is interesting that the mRNA produced by each of the three novel promoters displayed unique expression profiles when normal versus tumor human primary samples were examined; one mRNA was constitutively expressed, one mRNA was downregulated in the tumor sample, and one mRNA was highly upregulated in tumor RNA. Interestingly, one of the promoters that we cloned which displayed high-affinity binding in vivo was shown to be repressed, not activated, by E2F1. Although most E2F target genes studied to date are activated in response to overexpression of E2F1, it has been shown that the cyclin D1 promoter is also repressed by E2F1 ( 48). In addition, the recent microarray analysis by Muller et al. provided evidence that E2Fs can both activate and repress cellular genes, although their data did suggest that most E2F-mediated repression was indirect ( 31). Additional evidence supporting E2F-mediated repression of the ChET 8 promoter can be extrapolated from a recent study examining the cell cycle fluctuations of thousands of human mRNAs ( 4). We have extracted the expression profiles of E2F1 and KIAA0254, the mRNA driven by the ChET 8 promoter, from the published microarray data. Interestingly, ChET 8 mRNA levels are inversely related to E2F1 mRNA levels (data not shown). Collectively, these findings support a role for E2F1 in repression of the ChET 8 promoter. Further experiments need to be performed to characterize similarities and differences between the promoters which are directly activated and those which are directly repressed upon overexpression of E2F1. However, these observations suggest that the nature and context of the E2F binding site may influence the role that E2F plays in regulation of a promoter.

In summary, the data presented in this paper establish the basis for cloning novel promoters regulated by specific transcription factors through chromatin immunoprecipitation techniques. Our initial data suggest that the E2F consensus binding sequence may not account for all potential in vivo E2F targets, possibly due to the roles of interacting proteins within the cellular environment. Importantly, the possibility that E2F family members can regulate promoters that lack consensus binding sites may aid in the understanding of microarray studies which show that hundreds of mRNAs can respond to overexpression of E2Fs ( 16, 31). Also, we find it most interesting that the expression profiles of the genes identified by using this unbiased approach are quite different from the expression profiles of the previously characterized E2F target genes. Finally, of particular interest are ChET 9 and ChET 4. ChET 9 contains a consensus E2F binding site and shows high-affinity binding in vivo and in vitro. Interestingly, the KIAA0160 mRNA which is transcribed by ChET 9 is upregulated in two different tumor types. The protein encoded by the KIAA0160 mRNA has high homology to a Drosophila protein called Su(z)12. This protein was isolated as a suppressor of a mutation of the gene for zeste, a site-specific DNA binding transcription factor. Although no characterizations of Su(z)12 have been performed; another suppressor of zeste, Su(z)2, is known to be a locus-specific chromosome binding protein. Therefore, it is possible that KIAA0160 will be involved in transcriptional regulation. ChET 4, which shows high-affinity E2F in vivo binding but does not contain a consensus E2F site, is the promoter region for the beclin 1 gene, a putative tumor suppressor gene. The Beclin 1 protein is thought to effect the degradation of cellular proteins and has been shown to be significantly downregulated in human breast carcinomas ( 26). Our future studies will be focused on understanding the role of Beclin 1 and KIAA1060 in neoplastic transformation.

ACKNOWLEDGMENTS

This work was supported in part by Public Health Service grant CA45250 (to P.J.F.), CA07175 (an NCI Cancer Center Core grant), HG01696 and GM61503 (to M.Q.Z.), and training grant CA09681 (A.S.W.) from the National Institutes of Health.

We thank David Bentley for the RNA polymerase II antibody, Alexander Kel for exon 2 computer sequence analysis, Scott Eberhardy, Carrie Graveel, and Tadge Kanjo for RNA samples, Julie Wells for technical assistance, and members of the Farnham laboratory for helpful discussions.

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Use of Chromatin Immunoprecipitation To Clone Novel E2F Target Promoters | Molecular and Cellular Biology

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Volume 21 , Number 20 15 October 2001 HeLa cells were grown in 50% alpha minimal essential medium and 50% Joklik's medium with 5% supplemental calf serum (HyClone) and 1% penicillin-streptomycin (Gibco). The cells were grown to a density of 2 × 10 5 to 5 × 10 5 per ml before being harvested for cross-linking experiments.

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Development and Characterization of a Highly Sensitive NanoLuciferase-Based Immunoprecipitation System for the Detection of Anti-Influenza Virus HA Antibodies

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Development and Characterization of a Highly Sensitive NanoLuciferase-Based Immunoprecipitation System for the Detection of Anti-Influenza Virus HA Antibodies

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Honda, Tomoko, Sumiko Gomi, Daisuke Yamane, Fumihiko Yasui, Takuya Yamamoto, Tsubasa Munakata, and Yasushi Itoh et al. 2021. "Development And Characterization Of A Highly Sensitive Nanoluciferase-Based Immunoprecipitation System For The Detection Of Anti-Influenza Virus HA Antibodies". Msphere. doi:10.1128/msphere.01342-20.

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Volume 6 , Number 3
30 June 2021

INTRODUCTION

Antibodies (Abs) are produced by plasma cells in response to non-self-antigens such as pathogen-associated molecules. Thus, the titer of specific Abs is a key parameter in the medical diagnosis of infectious disease.

Influenza virus infection causes acute respiratory disease and imposes a heavy burden on society. Effects are particularly severe in young children and the elderly ( 1). Annually, seasonal influenza epidemics cause 3 to 5 million severe cases in the world ( 2). Hemagglutinin (HA) and neuraminidase (NA) proteins are the major surface antigens (Ags) of the influenza virus and are targets for vaccine and antiviral drugs, respectively ( 3-5). HA-specific binding Abs and neutralizing Abs induced by various vaccines mediate protection against virus invasion.

The HA inhibition (HI) assay, virus neutralization (NT) assay, and enzyme-linked immunosorbent assay (ELISA), are widely used for epidemiologic and immunologic studies of influenza virus as well as in the evaluation of vaccine efficacy ( 6). However, these assays have drawbacks: the HI assay requires pretreatment of sera to remove endogenous nonspecific inhibitors, the NT assay requires a relatively large amount of serum, and the ELISA is not suitable for quantitative analysis of Ab levels because of the assay's narrow dynamic range, given that the readout relies on absorbance. Moreover, preparation of Ags for ELISA is labor-intensive and expensive.

As an alternative to ELISA-based methods, the luciferase immunoprecipitation system (LIPS) was developed for the detection of Ag-specific Abs in sera from various animals ( 7, 8). LIPS requires the production of a protein comprising a reporter enzyme (luciferase) fused to an Ag of interest; this reagent is expressed from a plasmid vector in cultured cells. The luciferase-Ag fusion protein is recognized by Ag-specific Abs; the resulting Ag-Ab complexes are immobilized on protein A/G beads that recognize the fragment crystallizable (Fc) region of immunoglobulin G (IgG), and the Ag-specific Abs are quantified by measuring luminescence emitted by the luciferase-Ag fusion protein. This immunoassay represents a major improvement over ELISA technology in that LIPS is easily implemented to quantify the humoral response profile to various Ags in a universal format, providing a large amount of Ag while decreasing the time and effort needed to prepare highly purified Ags. Furthermore, LIPS has a low background with a broad dynamic range (spanning approximately 6 to 7 orders of magnitude). Luciferase-encoding genes derived from Gaussia princeps (copepod; GLuc), Renilla (jellyfish; RLuc), and Photinus pyralis (firefly; FLuc) have been employed for use in the LIPS assay ( 9-12). Among the luciferases, GLuc is in principle approximately 100-fold brighter than RLuc, although the former has a shorter half-life (about 2 min) ( 13). To overcome these issues, we adopted NanoLuciferase (NLuc) for use in the LIPS. Notably, NLuc is a small (19-kDa) enzyme derived from the deep-sea shrimp Oplophorus gracilirostris. The intensity of luminescence produced by NLuc is 150-fold higher than either FLuc or RLuc. Moreover, NLuc shows improved luminescence stability compared to the other luciferases, possessing a signal half-life of greater than 2 h ( 14).

HA proteins are known to assemble into a trimeric form via a process that depends on multiple posttranslational modifications, including disulfide bond formation and glycosylation ( 15). Several studies have shown that N-linked glycosylation of HA is important not only for the regulation of protein folding and stability but also for virus infectivity and antigenicity ( 16, 17). Immunogenicity of recombinant HA proteins is affected by both N-linked glycosylation and trimerization. Thus, the accurate measurement of HA-specific Abs requires the construction of recombinant HA proteins with native conformations.

In the present study, we developed recombinant influenza virus HA proteins fused to NLuc and used these fusion proteins for LIPS. We then employed the resulting assay, which we designated NanoLIPS, for monitoring the titer of Abs present in animal sera following either influenza virus infection or vaccination. Our data indicated that NLuc inserted at the C terminus of HA is modified by N-glycosylation and forms intact trimeric complexes that are recognized by monoclonal Abs (MAbs) that bind to the globular head or stalk domain of HA.

RESULTS

Supplemental Material
NLuc-based LIPS is more sensitive than GLuc-LIPS for detection of Abs in mouse sera.

GLuc fusion proteins typically have been employed for the LIPS assay (GLIP). To compare the sensitivity of GLuc and NLuc reporters for the LIPS assay, we generated recombinant constructs encoding influenza virus H5N1 HA protein (A/Bar-Headed Goose/Qinghai Lake/1A/05, clade 2.2) fused with either GLuc (GLuc-H5 HA) or NLuc (NLuc-H5 HA) at the C terminus of the HA protein ( Fig. 1A). Mouse sera were collected 3 weeks after vaccination with recombinant vaccinia virus expressing H5N1 HA protein (rDIs-H5 HA), and the titers of Ag-specific Abs were measured by either of the two different LIPS assays. NanoLIPS and GLIP were conducted as depicted schematically in Fig. 1B. To optimize conditions for diluting serum samples, we compared several blocking buffers from commercial sources. Unlike conventional blocking agents (e.g., bovine serum albumin [BSA] or skim milk) that yielded high background, Odyssey blocking buffer from LI-COR provided the highest signal-to-noise ratio ( Fig. 1C). As shown in Fig. 1D, the ratio of values obtained for sera from immunized mice compared to that from naive mice (using 1,000-fold dilutions, i.e., 1:1,000) was at least 20-fold higher when assayed by NLuc-H5 HA-based LIPS than by GLIP. Furthermore, NLuc signal was readily detectable even at 1:100,000 dilutions of sera, in contrast to the signal from GLuc-H5 HA, which appeared to plateau at dilutions of 1:30,000 ( Fig. 1D). No HA-specific Abs were detected from mice immunized with an empty vector control (DIs; Fig. 1D). We confirmed that the signal half-life of NLuc exceeded 150 min ( Fig. 1E), whereas the GLuc signal decreased much more rapidly, exhibiting a half-life of less than 1 min ( Fig. 1E). The greater stability of NLuc allowed us to establish an injector-free protocol for the measurement of luminescence. Together, these results demonstrate that this improved LIPS assay based on NLuc offers a highly sensitive and simple protocol for the detection of Ab titers.

FIG 1 Comparison between NanoLuciferase (NLuc)- and Gaussia luciferase (GLuc)-based luciferase immunoprecipitation system (LIPS) assays. (A) Schematic representation of the recombinant GLuc-HA or NLuc-HA constructs used for LIPS assays. (B) Schematic of the procedure used for the NanoLIPS and GLIP assays. (C) Comparison of serum dilution buffers from several commercial sources. Raw values of relative luciferase activity (top) and respective signal-to-noise ratio (bottom) are shown (n = 3). (D) Detection of HA-binding antibodies by LIPS assay based on NLuc-HA versus GLuc-HA. Sera were collected from mice 3 weeks after inoculation with recombinant vaccinia virus expressing HA (rDIs H5 HA) derived from H5N1 virus (A/Bar-Headed Goose/Qinghai Lake/1A/05). Sera from three different vaccinated mice (high, middle, and low antibody titers) were diluted 1:1,000 to 1:100,000 (n = 3). The panel shows P values only for the comparison between NLuc-HA- and GLuc-HA-based assays performed using sera from mice vaccinated with rDIs-H5 HA. With the exception of rDIs-H5 HA #3 at the 1:30,000 dilution, assayed via NanoLIPS based on NLuc-HA, sera from mice vaccinated with rDIs-H5 HA showed significantly higher signal-to-noise ratios than did sera from control mice (vaccinated with DIs). Statistical analyses were performed by two-tailed one-way ANOVA with post hoc Tukey's multiple-comparison tests (***, P < 0.001; ****, P < 0.0001). (E) Stability of NLuc versus GLuc signals. Raw luminescence activities (left), relative values (right), and the half-life (t 1/2) of each signal are shown. Values are shown as means ± standard deviations (SD) (n = 3).

For NanoLIPS, the NLuc fusion Ags were produced using HEK293FT cells. We expected that the quality of Ags might be affected by cell conditions. Therefore, to assess the batch-to-batch variability, we performed a NanoLIPS assay using three different batch lysates as Ags. Mouse sera were collected 3 weeks after inoculation with control DIs or rDIs-H5 HA and used to analyze the reproducibility of the NanoLIPS assay. No significant difference was observed in either the reactivity of H5 HA-specific Abs (see Fig. S1, left, in the supplemental material) or the background ( Fig. S1, right) among the three different lysate batches.

FIG S1

Effects of batch-to-batch variability in NanoLIPS antigen. NanoLIPS was performed using sera from mice 3 weeks after vaccination with rDIs-H5 HA (left) or control DIs (right). Statistical analysis was performed by two-tailed one-way ANOVA with post hoc Tukey's multiple-comparison tests. Values are plotted as means ± SD ( n = 3). ns, not significant ( P ≥ 0.05). Download FIG S1, EPS file, 1.3 MB.

Supplemental Material
C-NLuc HA is conformationally intact and undergoes glycosylation to form a trimer complex.

We next constructed HA proteins fused with NLuc at the HA C-terminal (C-NLuc-H5 HA) or N-terminal (N-NLuc-H5 HA) end to determine whether the position of the NLuc insertion affected the assay ( Fig. 2A). C-NLuc-H5 HA had a higher signal-to-noise ratio and better detection limit than N-NLuc-H5 HA when rDIs-H5 HA-vaccinated mouse sera were tested for HA-specific Abs ( Fig. 2B). Given that ELISA is most typically used for detecting HA-specific Abs, we analyzed the correlation between the results of NanoLIPS and ELISA using a recombinant HA protein (rH5 HA) purified from recombinant vaccinia virus rVV-HA-infected RK13 cells as Ag. Data from NanoLIPS based on C-NLuc-H5 HA ( r = 0.81, P < 0.0001), but not those from the assay based on N-NLuc-H5 HA ( r = −0.34, P = 0.16), exhibited significant correlation with the results from ELISA ( Fig. 2C). Furthermore, to determine the detection limit of each NanoLIPS method, serially diluted anti-rDIs-H5 HA serum samples were used for the assays. NanoLIPS demonstrated a broad linear range (from serum volume of 6.3 × 10 −2 to 6.3 × 10 −5 μl/reaction mixture, r ² = 0.99) ( Fig. 2D), in contrast to the ELISA-based method (in which values plateaued at dilutions as low as 1:100,000 [1:100 × 2 ¹⁰]) ( Fig. 2E). Thus, compared to conventional ELISA, NanoLIPS produced comparable results with a superior detection limit.

FIG 2 Comparison of effect of NLuc reporter inserted at the N terminus versus the C terminus of HA. (A) Schematic representation of N-terminal and C-terminal NLuc-HA constructs (N-NLuc-H5 HA and C-NLuc-H5 HA, respectively). (B) Detection of HA-binding antibodies by NanoLIPS based on N-NLuc- versus C-NLuc-H5 HA. Sera were collected from mice 3 weeks after inoculation with vaccinia virus expressing HA from H5N1 virus (A/Bar-Headed Goose/Qinghai Lake/1A/05) (n = 3). Comparisons were performed by two-tailed nonpaired Student's t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001 versus N-NLuc-H5 HA). (C) Correlation between ELISA and NanoLIPS assays. ELISA detection of HA-binding antibodies in mouse sera was conducted using C-terminally His-tagged recombinant H5 HA (A/Bar-Headed Goose/Qinghai Lake/1A/05). NanoLIPS assays based on N-NLuc-H5 HA or C-NLuc-H5 HA were performed using the same sera (n = 18). Abs, absorbance. (D and E) Detection limit of C-NLuc-H5 HA-based NanoLIPS versus ELISA. Sera from rDIs-H5 HA-vaccinated mice were subjected to serial dilution as indicated below the panels; assays were performed in triplicate (n = 3). OD, optical density. (F) Glycosylation of C-NLuc-H5 HA and N-NLuc-H5 HA was tested by digestion with peptide-N-glycosidase F (PNGase F) and electrophoresis by SDS-PAGE under reducing conditions. (G) Trimer complex formation by C-NLuc-H5 HA. Lysates of cells expressing C-NLuc-H5 HA, N-NLuc-H5 HA, or recombinant His-tagged H5N1 HA protein were electrophoresed by SDS-PAGE under reducing or nonreducing conditions and detected using anti-H5 HA 14A7 monoclonal antibody. (H) Schematic representation of N-glycosylation or trimerization conditions of each NLuc-tagged HA. (I and J) Monoclonal antibody against globular head domain of H5 HA (I) or stalk domain (J) of HA were detected by NanoLIPS based on N-NLuc-H5 HA versus C-NLuc-H5 HA, or C-NLuc-H1 HA (n = 3). ****, P < 0.0001; ns, not significant (P ≥ 0.05). (K) Verification of HA specificity of the NanoLIPS assay. Mice were inoculated with either rDIs-H5 HA or rDIs-H1-HA (derived from H1N1 virus). Four weeks after vaccination, mouse sera were collected and used for measuring HA-specific antibodies by NanoLIPS assay based on C-NLuc-HA derived from homologous or heterologous serotype HA (C-NLuc-H1HA or C-NLuc-H5 HA). Values are shown as means ± SD (n = 3).

N-linked oligosaccharides on the HA protein play important roles in virus antigenicity, receptor binding, and infectivity ( 18, 19). Several reports have indicated that the sites of N-linked glycosylation within the HA stalk domain are well conserved among influenza virus strains and that glycosylation of HA contributes to the structural stability of the HA trimer ( 20). Therefore, we examined whether NLuc-HA proteins undergo the expected posttranslational modification and functional trimer complex formation under native conditions. Glycan attached to the globular head of HA is important for virus antigenicity ( 21); however, only C-NLuc-H5 HA (and not N-NLuc-H5 HA) exhibited glycosylation, as revealed by sensitivity to peptide-N-glycosidase F (PNGase F) treatment ( Fig. 2F). Furthermore, native PAGE revealed that C-NLuc-H5 HA formed a trimer complex, as also seen with recombinant HA protein (rH5 HA) ( Fig. 2G). Thus, these data indicated that the insertion of NLuc at the C terminus of HA does not impair the glycosylation profile or functional conformation of HA protein ( Fig. 2H).

Next, we examined the binding of N- or C-NLuc-HA by MAbs that recognize the globular head or stalk domain of HA; this analysis used four different Abs known to target distinct HA epitopes or domains. Escape mutant analysis has indicated that MAbs 14A7 and 3B9 bind near the R156 and T183 residues, respectively, both of which are located in the globular head domain of H5 HA ( 22). CR9114 has been reported to bind an epitope near the hydrophobic pocket in HA2, thereby neutralizing both group 1 and group 2 influenza A viruses ( 23). CR6261 binds near the highly conserved stalk domain of group 1 influenza A virus ( 24). As shown in Fig. 2I and J, C-NLuc-H5 HA yielded significantly higher NLuc signal than N-NLuc-HA following immunoprecipitation with MAbs against either the globular head (14A7, 3B9) or the stalk domains (CR9114, CR6261) of HA. Similarly, C-NLuc-H1 HA showed comparable levels of NLuc signal following immunoprecipitation with MAbs against the stalk domain of HA but not with MAbs against the globular head domain of H5 HA. Furthermore, we compared the sensitivities of the C-NLuc-HA-based NanoLIPS assay and an ELISA that used the CR9114 MAb. NanoLIPS was able to detect 3 ng of Ab ( Fig. S2A, left), a sensitivity that matched that of the ELISA ( Fig. S2A, right). Both assays showed a significant correlation ( Fig. S2B, r = 0.998, P < 0.0001). These data indicated that C-NLuc-HA has an intact conformation and can be used for detecting Abs that bind to the globular head domain of HA and also for detecting Abs that bind to the stalk domain of HA.

FIG S2

Comparison of the sensitivities of the C-NLuc-HA-based NanoLIPS assay and an ELISA. (A) NanoLIPS (left) and ELISA (right) were performed using serial dilutions of the CR9114 monoclonal antibody (MAb). ELISA was conducted using C-terminally His-tagged recombinant H5 HA (A/Bar-Headed Goose/Qinghai Lake/1A/05) ( n = 3). (B) ELISA and NanoLIPS assay were performed using serial dilutions of the CR9114 MAb. Linear regression curves were fitted to the data sets. Pearson's correlation coefficient (r) and a two-tailed P value were calculated for the combination of ELISA and NanoLIPS data sets. Download FIG S2, EPS file, 1.2 MB.

However, influenza A viruses have at least 18 different subtypes of HA, meaning that a HA-specific Ab detection system would be required to distinguish among these HA serotypes. Therefore, we examined whether NanoLIPS could differentiate specific HAs from other influenza subtypes. NanoLIPS based on C-NLuc-H5 HA detected Abs from the sera of mice vaccinated with rDIs-H5 HA, whereas no HA-specific Abs were detected in the sera from rDIs-H1-vaccinated mice ( Fig. 2K). Similar results were obtained using C-NLuc-H1 HA as an Ag ( Fig. 2K). Thus, both NanoLIPS Ags could be immunoprecipitated only with the respective subtype-specific antisera. These results indicated that NanoLIPS can distinguish between H5 HA (avian HA) and H1 HA (seasonal HA).

Supplemental Material
NanoLIPS assay can be used for detection of Abs from cynomolgus macaque and tree shrew.

Various experimental animal models have been developed for influenza challenge experiments ( 25-28). To evaluate whether NanoLIPS assay can be employed in these different models, we performed NanoLIPS assays using sera from two different species, cynomolgus macaque (Macaca fascicularis) and tree shrew (Tupaia belangeri). Macaque models have been used for investigating influenza pathogenesis and for evaluating the protective efficacy of influenza vaccines ( 29, 30). In the present study, macaques were immunized by two injections (administered 3 weeks apart) of 1 × 10 ⁷ PFU per injection of vaccinia virus (rDIs-H5 HA), followed by challenge with influenza virus 2 weeks after the booster ( Fig. 3A). Ab responses against H5 subtype HA protein were measured using the NanoLIPS assay. After the first vaccination, the titer of HA-specific Abs increased gradually; the titer then increased approximately fourfold following the second vaccination (the boost, administered at 3 weeks) ( Fig. 3B). Following the challenge with H5N1 highly pathogenic avian influenza virus (HPAIV), a large further increase (about 10-fold) in H5 HA-specific Ab titer was detected ( Fig. 3C). To verify whether NanoLIPS is usable for detecting Ag-specific Ab responses in this animal model, we also investigated H5 HA-specific Ab responses by ELISA, using the same sera as those tested used in NanoLIPS; the ELISA employed recombinant H5 subtype HA protein as Ag. The data from NanoLIPS based on C-NLuc-H5 HA ( Fig. 3B and C) exhibited a significant positive correlation with those from ELISA ( Fig. S3A [ r = 0.94, P < 0.0001] and S3B [ r = 0.94, P < 0.0001]).

FIG 3 Application of NanoLIPS to detect HA antibodies in sera from macaques or tree shrews. (A) Schematic representation of the study design. Macaques were immunized intradermally (on the arm) with rDIs-H5 HA at 1.0 × 10⁷ PFU/animal at 0 weeks and boosted 3 weeks later at the same dose. Serum samples were collected every week after immunization. Five weeks after the initial vaccination, macaques were infected with 3.0 × 10⁶ PFU/animal of highly pathogenic avian influenza virus (HPAIV) A/whooper swan/Hokkaido/1/2008 H5N1. (B) Detection of HA antibodies by NanoLIPS based on C-NLuc-H5 HA at different time points postimmunization (n = 3). (C) NanoLIPS detection of HA antibodies in sera from macaques at 7 days after infection (dpi) with influenza virus. Statistical analysis was performed by two-tailed one-way ANOVA with post hoc Dunnett's multiple-comparison tests (****, P < 0.0001 versus 0 dpi). (D) Schematic representation of the study design. Tree shrews were infected with 10⁶ PFU/animal of A/VietNam/UT3040/2004 H5N1 virus. Seven days after infection, tree shrews were euthanized, and serum samples were collected. (E) Specific antibodies measured by NanoLIPS assay using C-NLuc-H5 HA as antigen (n = 3). Statistical analysis was performed by two-tailed nonpaired Student's t test (***, P < 0.001; ****, P < 0.0001). Values are shown as means ± SD.

FIG S3

Correlation analysis between ELISA and NanoLIPS. The data for NanoLIPS are from Fig. 3B (A) and Fig. 3C (B). Detection of H5 HA-specific antibodies by ELISA was performed using the same sera as those used for detection by NanoLIPS. ELISA was conducted using C-terminally His-tagged recombinant H5 HA (A/Bar-Headed Goose/Qinghai Lake/1A/05). Linear regression curves were fitted to the data sets. Pearson's correlation coefficients (r) and two-tailed P values were calculated for the indicated combinations of data sets. Download FIG S3, EPS file, 1.2 MB.

The tree shrew is a small mammal that shares genetic similarities with primates ( 31, 32). We and other groups have demonstrated that tree shrews are susceptible to infection with both seasonal and avian influenza viruses ( 33-35). In the present study, serum samples from tree shrews infected with H5N1 influenza virus were collected 7 days after infection ( Fig. 3D). Again, increases (fivefold or greater) in HA-specific Abs were detected in virus-infected tree shrew using a NanoLIPS assay based on C-NLuc-H5 HA ( Fig. 3E). These results demonstrated that NanoLIPS can be used for detecting Ag-specific Abs in both macaque and tree shrew models.

Evaluation of vaccine efficacies using NanoLIPS.

We next examined whether NanoLIPS can be used for testing vaccine efficacy. We evaluated the in vivo efficacy of rDIs-H5 HA against H5N1 HPAIV infection in a murine lethal infection model. Mice vaccinated with rDIs-H5 HA were infected with a lethal dose of H5N1 HPAIV and observed for body weight changes ( Fig. 4A). All rDIs-H5 HA-vaccinated mice exhibited transient mild weight loss (a mean 9% [compared to baseline] at 3 days postinfection [dpi]), but these animals recovered rapidly, and all survived. In contrast, severe weight loss (>30% compared to baseline) and subsequent mortality (humane sacrifice) was seen in all control vaccinated mice by 10 dpi ( Fig. 4B and C). We next evaluated correlations between the protective effect of vaccination and H5 HA-specific Ab titers (3 weeks after vaccination), as measured by NanoLIPS using C-NLuc-H5 HA; relative body weight (percentage of weight on day 0) was used as an indicator of vaccine efficacy. H5 HA-specific Ab responses were analyzed in antisera from the vaccinated mice by NanoLIPS assay and ELISA. As shown in Fig. 4D, the results obtained by NanoLIPS based on C-NLuc-H5 HA exhibited a significant positive correlation with the results obtained by ELISA ( Fig. 4D, r = 0.81, P < 0.0001). We then quantified the H5 HA-specific Ab concentration based on the relative light unit (RLU; where one unit is defined as the number of luminometer units measured in 1 second) obtained from the NanoLIPS assay. First, the calibration curve was determined using CR9114, which is a commercially available and well-characterized H5 HA-specific MAb ( K D = 0.9 nM against H5 HA derived from A/Vietnam/1203/2004 H5) ( 23), which gave the linear regression ( r ² = 0.99, y = 2839.6 x + 2355.8) ( Fig. 4E). The H5 HA-specific Ab concentration in antisera of vaccinated mice was then calculated by fitting the data to the linear equation. Notably, a correlation was detected between Ab concentration detected by C-NLuc-H5 HA-based NanoLIPS and relative body weight ( P < 0.0001) ( Fig. 4F). These results demonstrated that NanoLIPS using the C-NLuc-fused H5 HA protein is expected to be of use for evaluating vaccine efficacy.

FIG 4 Correlation between NLuc activity and vaccine efficacy. (A) Schematic representation of the study design. Mice were immunized intradermally with rDIs-H5 HA vaccine at 10⁷ PFU/animal; serum samples were collected 3 weeks after vaccination. Four weeks after vaccination, mice were infected intranasally with 10⁴ PFU/animal of highly pathogenic avian influenza virus (HPAIV) A/whooper swan/Hokkaido/1/2008 H5N1. (B and C) Body weight change (B) and survival rate (C) were monitored following influenza virus challenge (for DIs, n = 3; for rDIs-H5 HA, n = 18). Statistical analysis was performed by two-tailed nonpaired Student's t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 versus DIs control). Values are shown as means ± SD. (D) Correlation between ELISA and NanoLIPS. The data for NanoLIPS are from panel F. Detection of H5 HA-specific antibodies by ELISA was performed using the same sera as those assessed by NanoLIPS. ELISA was conducted using C-terminally His-tagged recombinant H5 HA (A/Bar-Headed Goose/Qinghai Lake/1A/05). Linear regression curves were fitted to the data sets, and Pearson's correlation coefficients and two-tailed P values were calculated for the indicated combinations of data sets. Absb, absorbance. (E) The calibration curve of NanoLIPS was determined using serial dilutions of the CR9114 MAb. A linear regression curve was fitted to the data set. The coefficient of determination (r ²) was calculated. (F) Correlation between relative body weight (3 days postinfection) and Ab concentration (3 weeks after vaccination). The NanoLuciferase activity has conversed to the Ab concentration based on the above linear regression curve (panel E). For DIs, n = 3 (open circles); for rDIs-H5 HA, n = 18 (closed squares). Linear regression curve was fitted to the data sets. Pearson's correlation coefficient (r) and a two-tailed P value were calculated.

DISCUSSION

This study demonstrated that NanoLIPS offers a highly sensitive and simple technique for the detection of Ag-specific Abs present in sera from mouse, macaque, and tree shrew. In recent years, LIPS has been widely used for assessing immune responses in infectious diseases caused by HIV ( 36), Ebola virus ( 37), and influenza virus ( 38). In a previous study, a binding Ab titer against HA-1 of influenza virus, measured via Renilla luciferase-based LIPS, demonstrated a positive correlation with HI-determined titer. Moreover, LIPS could detect Ab titers that the HI assay could not detect ( 39). Thus, LIPS represents a powerful method for measuring Abs associated with various viral infections or vaccinations.

We further showed that NanoLIPS provides exceptional sensitivity compared to GLuc-based LIPS, allowing us to detect very low levels of HA-binding Abs in small aliquots of sera, under conditions in which the previous methods were unable to detect such Abs. Luminescence catalyzed by NLuc was at least 20-fold brighter than that from GLuc and exceptionally stable (half-life > 155 min), bypassing the need to use injector equipment for the Luc measurement. Moreover, NanoLIPS uses cell lysates expressing recombinant proteins as Ags. Because this technique bypasses the necessity of the purification step, NanoLIPS is a relatively simple method to generate Ags. The use of mammalian cells to express recombinant proteins allows for the generation of more suitable Ags that have undergone appropriate posttranslational modifications such as glycosylation.

We also showed that the NLuc fusion protein used for NanoLIPS had to be designed with caution, given that the antigenicity of the target protein was significantly affected by the position (N terminal versus C terminal) of the NLuc fusion. Careful assessment of NLuc positions at either the N or C terminus demonstrated that NanoLIPS based on C-NLuc, but not that based on N-NLuc, was capable of detecting MAbs that bind the globular head and stalk domains of influenza virus HA ( Fig. 2I and J). We further found that C-terminal NLuc retains the intact conformation of HA, undergoing normal glycosylation and trimer formation, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE analysis ( Fig. 2F and G). NanoLIPS results based on C-NLuc-H5 HA yielded data that exhibited a significant correlation with the protective effects of an rDIs-H5 HA vaccine ( Fig. 4F). When vaccinated with rDIs-H5 HA, recipients produce a variety of Abs that bind to the globular head domain or stalk domain of HA. Among the strategies for influenza virus vaccine development, the stimulation of humoral responses against the globular head domain of HA is an important approach to preventing virus binding to the host cell or membrane fusion. On the other hand, Abs against the stalk domain of HA induce broad immunity and prevent infection with heterosubtypic influenza viruses ( 40). Thus, the induction of both types of Abs is crucial for preventing influenza virus infection. C-NLuc-H5 HA-based NanoLIPS was able to react with both Abs with specificity for the globular head domain of HA and those with specificity for the stalk domain. As shown in Fig. 2I and J, the binding sites and cross-reactivities of the detected Abs can be inferred using several serotypes of C-NLuc-HA as bait. Notably, the use of chimeric HAs (i.e., HAs comprising the head and stalk domains from distinct groups) ( 41) fused at their C termini with NLuc is expected to be very useful for binding site prediction of the detected Abs. Therefore, NanoLIPS, which facilitates Ag preparation, is expected to facilitate the evaluation of vaccine efficacy, supplementing or replacing the use of conventional methods such as ELISA and HI assay. It will be of great interest to test whether NanoLIPS can discriminate between Abs that recognize the globular head or the stalk domain of HA, potentially permitting more accurate analysis of the correlation of domain recognition and protective efficacy.

In the field of infectious disease, a variety of animal models are used. Because some viruses have a narrow host range, multiple animal models must be used in experiments. For instance, the hepatitis B virus exclusively infects humans, tree shrews, and chimpanzees ( 42, 43). Thus, an ideal immunological analysis should be applicable in a diverse range of animals. In the present study, we demonstrated that NanoLIPS can be used to detect Abs in serum samples derived from various experimental animal models, including the mouse, macaque, and tree shrew. Like other LIPS assays, NanoLIPS requires (in principle) only NLuc-tagged Ag and protein G beads for trapping the IgG present in small aliquots of sera. Thus, this assay system should be applicable for serum samples derived from various animal species, including wild animals, so long as the species' immunoglobulins have affinity permitting binding to protein G. Further experiments will be necessary to evaluate the effectiveness of NanoLIPS in other experimental animals.

In this LIPS method, protein G magnetic beads do not permit distinction among specific classes of immunoglobulins. During actual immunization or viral infection, IgM-type Abs are produced in the primary Ab response and then undergo class switching to produce IgA or IgG Abs. Thus, the detection of IgM is often considered an indicator of recent infection, whereas the detection of IgG indicates previous exposure to an Ag or pathogen. Future work will be needed to establish immunoglobulin class-specific measurement methods.

Our data demonstrated that the NanoLIPS assay provides a simple technique for preparation of the assay Ag, yields data with a broad dynamic range, is highly sensitive, and is amenable to high-throughput analysis. This assay may be useful as a diagnostic tool for monitoring the history of infection and the effects of vaccination.

MATERIALS AND METHODS

Ethics statement.

This study was carried out in strict accordance with the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science ( 44) and the recommendations in the Guide for the Care and Use of Laboratory Animals ( 45). All animal care and experimental procedures were performed according to the guidelines established by the Tokyo Metropolitan Institute of Medical Science Subcommittee on Laboratory Animal Care. All protocols were approved by the Committee on the Ethics of Animal Experiments of the Tokyo Metropolitan Institute of Medical Science.

Generation of NLuc-antigen fusion-encoding constructs.

To construct NLuc-expressing vectors (pCAGGS-N-NLuc or pCAGGS-C-NLuc), the NLuc-encoding gene was amplified by PCR using Q5 polymerase (New England Biolabs, Ipswich, MA) with gene-specific primer sets; the pNL1.1 vector (Promega, Madison, WI) was used as the template for the NLuc-encoding sequences. The amplicons were digested with AsiSI and SbfI and were ligated into AsiSI-SbfI double-digested pCAGGS-Neo (Wako, Osaka, Japan) in which the simian virus 40 (SV40) poly(A) sequence had been replaced by the rabbit β-globin poly(A). Next, the gene for influenza virus HA (A/Bar-Headed Goose/Qinghai Lake/1A/2005 [H5N1-Clade2.2]) was amplified by PCR using gene-specific primers. The pCAGGS-N-NLuc and pCAGGS-C-NLuc vectors were linearized with restriction enzyme AsiSI or SbfI, respectively. The HA-encoding PCR fragments then were cloned into each vector using an In-Fusion HD Cloning kit (TaKaRa Bio, Shiga, Japan), yielding in-frame fusions of the NLuc- and HA-encoding open reading frames (ORFs).

Production of GLuc or NLuc fusion proteins.

To express influenza virus HA fused to GLuc or NLuc, HEK293FT cells (ATCC, Manassas, VA) maintained in Dulbecco's modified Eagle medium (Gibco) containing 10% fatal bovine serum (FBS) were transfected with purified plasmid DNA using Polyethylenimine MAX (Polysciences, Inc., Warrington, PA); the transfection was performed using 70 to 90% confluent cells grown in six-well plates. Twenty-four hours after transfection, cells were rinsed with phosphate-buffered saline without CaCl ₂ and MgCl ₂ [PBS(−)], and chilled Renilla lysis buffer (Promega) was distributed at 200 μl/well. Cells then were harvested with a cell scraper, and the lysate from each well was transferred to a 1.5-ml microfuge tube and centrifuged at 13,000 ×  g for 10 min at 4°C. The resulting supernatant was transferred to a fresh tube and stored at −80°C until use.

Western blot analysis.

To analyze N-linked glycosylation of HA proteins, cell lysates containing NLuc-fused proteins were treated for 1 h at 37°C with peptide-N-glycosidase F (PNGase-F) (catalog no. P0704S; New England Biolabs, Inc.) and then separated by SDS-PAGE (10 μg total protein) using a SuperSep Ace 5 to 20% gel (Wako, Osaka, Japan) under reducing conditions, and the resulting gel was transferred to a polyvinylidene difluoride membrane. After blocking with Odyssey blocking buffer (LI-COR, Lincoln, NE), the membranes were incubated for 16 h at 4°C with rabbit anti-H5 HA polyclonal Ab (pep10; raised in our laboratory against a H5N1 HA peptide with the sequence DAAEQTRLYQNPTTYISVG) diluted 1:1,000 in the blocking buffer. After the membranes were washed three times with Tris-buffered saline containing 0.05% Tween 20 (TBS-T), they were incubated for 1 h at room temperature with anti-rabbit IgG IRDye 800CW (LI-COR) diluted 1:20,000 with the blocking buffer. After the membranes were washed three times with TBS-T, they were visualized using the Odyssey CLx infrared imaging system (LI-COR). Native PAGE was used to detect trimer complex formation by the HA proteins as described previously ( 46). Briefly, lysates suspended in native sample buffer (62.5 mM Tris-HCl [pH 6.8], 15% glycerol) were separated in running buffer (25 mM Tris and 192 mM glycine [pH 8.4] with or without 1% deoxycholate in the cathode and anode chambers, respectively). Mouse anti-H5 HA 14A7 MAb (developed in our laboratory) ( 22) and anti-mouse IgG IRDye 680RD (LI-COR) were used for detection of proteins separated by native PAGE.

LIPS assay.

To optimize conditions for serum dilution, we compared several blocking buffers. The commercial blocking buffers tested included the following: Block Ace (catalog no. UKB80; Sumitomo Dainippon Pharma Co., Ltd. [formerly DS PHARMA Biomedical], Osaka, Japan), Blok FL (catalog no. WBAVDFL01; Millipore, USA, Burlington, MA), Can Get Signal-1 (catalog no. NKB-101S; Toyobo, Osaka, Japan), h-Block-w (catalog no. BCL-BKHW-01; Beacle, Kyoto, Japan), Immuno enhancer A (catalog no. 294-68601; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), Intercept (catalog no. 927-70001; LI-COR), and Odyssey blocking buffer (catalog no. 927-40000; LI-COR). NLuc or GLuc fusion proteins were diluted to 2 × 10 ⁸ RLU (where one unit is defined as the number of luminometer units measured in 1 second) per ml with Tris-based buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM MgCl ₂, 1% Triton X-100, 25% glycerol) and transferred to a 96-well white plate (catalog no. 3789A; Corning, Corning, NY) at 50 μl (10 ⁷ RLU) per well. Mouse, tree shrew, or macaque sera diluted at 1:100 or 1:1,000 with Odyssey blocking buffer were added to the wells, and the plate was incubated for 1 h at room temperature with continuous rotation. Subsequently, 1 μl of SureBeads protein G magnetic beads (Bio-Rad, Hercules, CA) was added to each well, and the plate was further incubated for 1 h at room temperature with continuous rotation. The beads in each well were washed four times with PBS(+) [PBS(−) containing 0.9 mM CaCl ₂ and 0.5 mM MgCl ₂] -Tween 20 [PBS(+) containing 0.5% Tween 20] and once with PBS(+), and then suspended in 10 μl of PBS(+). An aliquot (10 μl) of Nano-Glo luciferase assay reagent (Promega) was added to each well, and the contents were mixed by pipetting. After 2 min of incubation at room temperature, the luciferase intensity was measured using an EnVision plate reader (PerkinElmer, Waltham, MA).

Enzyme-linked immunosorbent assay (ELISA).

Ninety-six-well plates were precoated with 0.2 μg/ml recombinant C-terminal His-tagged H5 HA (derived from H5N1 A/Bar-Headed Goose/Qinghai Lake/1A/2005) protein in 50 mM carbonate buffer for 18 h at 4°C and then treated with blocking buffer (PBS containing 1% bovine serum albumin [BSA], 0.5% Tween 20, and 2.5 mM EDTA) for 2 h at room temperature. Diluted mouse sera were added to the wells, and the plates were incubated for 18 h at 4°C. After the wells were washed three times with PBS(−)-T [PBS(−) containing 0.05% Tween 20], horseradish peroxidase-conjugated anti-mouse IgG Ab (1:8,000) was distributed to the individual wells, and the plates were incubated for 1 h at room temperature. After the wells were washed five times, they were incubated with o-phenylenediamine for 30 min. The reactions were quenched by the addition of 2 M sulfuric acid, and the absorbance at 492 nm was measured using an EnVision plate reader.

Generation of recombinant vaccinia virus.

Synthetic DNAs encoding the multibasic-site-deleted HA protein of H5N1 HPAIV (A/Bar-Headed Goose/Qinghai Lake/1A/05 [clade 2.2]) were purchased from Sloning BioTechnology (Puchheim, Germany). DNAs were amplified by PCR using specific primers with flanking restriction enzyme sites (HindIII). The resulting PCR fragments were digested with HindIII and ligated into a plasmid for recombination with the attenuated vaccine virus DIs strain (pUC/DIs-HA). pUC/DIs-HA was linearized with restriction enzymes and transfected into primary chicken embryo fibroblasts that had been infected with vaccinia virus strain DIs at a multiplicity of infection of 10 ( 47). After 24 h, the virus-cell mixtures were harvested and frozen at −80°C until required. Taking advantage of the fact that influenza virus HA protein agglutinates with guinea pig erythrocytes but vaccinia virus HA protein does not, HA-positive recombinant viruses that can recognize guinea pig erythrocytes were purified three times by plaque cloning, and the resulting virus stock was designated rDIs-H5 HA. Similarly, rDIs-H1 HA was generated by inserting HA gene derived from H1N1 pandemic influenza virus (A/California/07/2009) into the genome of strain DIs.

Vaccination, infection, and serum sampling.

BALB/c mice (7-week-old females) were purchased from SLC (Hamamatsu, Japan). Mice were inoculated once with 10 ⁷ PFU per animal of rDIs-HA or DIs-empty (DIs); the latter served as controls. The inoculum was administered by intradermal (i.d.) injection at 10 sites of injection (5 μl/site) distributed across the lower back. Sera were collected from peripheral blood 3 weeks after vaccination. Four weeks after vaccination, mice were infected with 1.0 × 10 ⁴ PFU/animal of HPAIV (A/whooper swan/Hokkaido/1/08 [H5N1-Clade 2.3.2.1]). The body weights of mice were monitored, and the loss of 30% initial body weight was defined as the endpoint for euthanasia. Cynomolgus macaques (Macaca fascicularis) were obtained from Ina Research (Nagano, Japan). Macaques were inoculated i.d. by two separate injections with 1.0 × 10 ⁷ PFU/injection of rDIs-HA or DIs, administered 3 weeks apart. Sera were collected once weekly after vaccination. Five weeks after the initial inoculation (2 weeks after the second inoculation), macaques were infected with 3.0 × 10 ⁶ PFU/animal of HPAIV (A/whooper swan/Hokkaido/1/08 [H5N1-Clade2.3.2.1]); sera were collected at 7 dpi. Northern tree shrews (Tupaia belangeri) were purchased from the Kunming Institute of Zoology, Chinese Academy of Sciences (Kunming, China). The tree shrews were bred in the animal facilities of the Kagoshima University and Tsukuba Primate Research Center for experimental use. Tree shrews were infected with 1.0 × 10 ⁶ PFU/animal of influenza virus (A/VietNam/UT3040/2004 [H5N1-Clade1]); sera were collected at 7 dpi.

Expression and purification of His-tagged recombinant HA.

The recombinant vaccinia virus expressed the multibasic-cyte-deleted HA protein derived from HPAIV A/Bar-Headed Goose/Qinghai Lake/1A/05 [H5N1-Clade2.2] (rVV-H5 HA). To express recombinant His-tagged influenza virus HA protein, RK13 cells (ATCC) maintained in minimum essential medium containing 5% FBS were infected with the rVV-H5 HA. Twenty-four hours after infection, cells were harvested with a cell scraper and stored at −80°C until use. Expressed His-tagged recombinant HA protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol.

Statistical analysis.

Statistical analyses were performed with Prism software (version 8.3.0; GraphPad, San Diego, CA). Results are reported as means ± standard deviations (SD). The P values were calculated using two-tailed nonpaired Student's t tests for data from two groups or using one-way analysis of variance (ANOVA) or two-way ANOVA followed by post hoc Dunnett's or Tukey's multiple-comparison tests, as appropriate, for data from more than two groups/variables. P values of less than 0.05 were considered significant. The correlation coefficient ( r value) was calculated using the corresponding function in Prism.

ACKNOWLEDGMENTS

We express our gratitude to Satoshi Naganawa for technical assistance with the recombinant vaccinia virus experiments.

This study was supported by grants from the JSPS KAKENHI (grant 15H04739) and the Tokyo Metropolitan Government, Japan.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We declare that we have no competing financial or nonfinancial interests.

T.H. and M.K. designed the study. S.G., D.Y., F.Y., T.Y., T.S., T.M., K.Y., Y.Y., K.T.-K., and M.K. performed the experiments. T.H., D.Y., F.Y., and M.K. analyzed the data. T.H., D.Y., F.Y., and M.K. wrote the manuscript. All authors reviewed the manuscript.

Information & Contributors

Information

Published In

Volume 6 * Number 3 * 30 June 2021

Editor: Marcela F. Pasetti, University of Maryland School of Medicine

History

Published online: 12 May 2021

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KEYWORDS

1. NanoLuciferase (NLuc)

2. broad dynamic range

3. cynomolgus macaque

4. influenza virus hemagglutinin (HA) protein

5. luciferase immunoprecipitation system (LIPS)

6. mouse

7. tree shrew

8. trimeric complex

Contributors

Authors

Division of Pathogenesis and Disease Regulation, Department of Pathology, Shiga University of Medical Science, Setatsukinowa, Otsu, Shiga, Japan

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan

Editor

University of Maryland School of Medicine

# data[].attributes.attachment_link.title
Development and Characterization of a Highly Sensitive NanoLuciferase-Based Immunoprecipitation System for the Detection of Anti-Influenza Virus HA Antibodies | mSphere

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Volume 6 , Number 3 30 June 2021 Antibodies (Abs) are produced by plasma cells in response to non-self-antigens such as pathogen-associated molecules. Thus, the titer of specific Abs is a key parameter in the medical diagnosis of infectious disease. Influenza virus infection causes acute respiratory disease and imposes a heavy burden on society.

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Gooderham, K. (n.d.). Transfer Techniques in Protein Blotting. Proteins, 165–178. doi:10.1385/0-89603-062-8:165

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Abstract

Polyacrylamide gel electrophoresis is an extremely powerful tool for the analysis of complex protein mixtures. Although the value of this method cannot be questioned, it is restricted in that the separated proteins remain buried within the dense gel matrix and are not readily available for further investigation. A number of methods have been developed in order to try and overcome this problem, for example the elution of proteins from excised gel slices (see Chapter 19 ). Alternatively, proteins have been studied while they are still buried within the gel using a variety of in situ peptide mapping (see Chapter 22 ) and gel overlay techniques (for example, see ref. 1). Unfortunately all of these methods have serious drawbacks: in the case of protein elution and in situ peptide mapping techniques, the resolution and number of bands that can be processed is restricted, whereas the gel overlay techniques are generally time-consuming and insensitive.

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