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Background on DFT and also uses and how it is used to diagnose conditions.

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The dFA test is based on the observation that animals infected by rabies virus have rabies virus proteins (antigen) present in their tissues. Because rabies is present in nervous tissue (and not blood like many other viruses), the ideal tissue to test for rabies antigen is brain. The most important part of a dFA test is flouresecently-labeled anti-rabies antibody. When labeled antibody is incubated with rabies-suspect brain tissue, it will bind to rabies antigen. Unbound antibody can be washed away and areas where antigen is present can be visualized as fluorescent-apple-green areas using a fluorescence microscope. If rabies virus is absent there will be no staining.

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rabies; dfa test; direct fluorescent antibody test; rabies virus proteins; antigen; nucleoprotein

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CDC - Diagnosis: Direct Fluorescent Antibody Test - Rabies

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The dFA test is based on the observation that animals infected by rabies virus have rabies virus proteins (antigen) present in their tissues. Because rabies is present in nervous tissue (and not blood like many other viruses), the ideal tissue to test for rabies antigen is brain.

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New method of acne disease fluorescent diagnostics in natural and fluorescent light and treatment control 

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New method of acne disease fluorescent diagnostics in natural and fluorescent light and treatment control 

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insight in how fluorescent markers are used to diagnose severe types of skin acne.

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New method of acne disease fluorescent diagnostics in natural and fluorescent light and treatment control

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How fluorescent isotopes bind to antibodies and the ways in which it is done

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How fluorescent isotopes bind to antibodies and the ways in which it is done

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using primary and secondary techniques to bind to the antibody.

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Rockland visualizes biological samples by using conjugated antibodies and a fluorescent microscope to localize cell structure, analyze delivery of proteins.

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different fluorescent radioactive markers 

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different fluorescent radioactive markers 

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The different markers used in biological procedures for diagnostics and there half life's

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Abstract

Introduction

The green fluorescent protein (GFP) from the jellyfish Aequoria victoria provides a visual marker that can be used in a wide variety of organisms ( Chalfie et al., 1994; Tsien, 1998). The wild-type protein has been significantly improved for use in mammalian cells by the incorporation of mutations such as S65T and F64L, which change the absorption spectrum and increase the rate of fluorophore formation ( Heim et al., 1995) and by `humanizing' the codon usage ( Zolotukhin et al., 1996). GFP folds into a unique, compact structure, the β-can ( Ormö et al., 1996; Yang et al., 1996), that is strongly resistant to chemical denaturation ( Ward and Bokman, 1982) and is observed to be stable in most cells ( Cubitt et al., 1995). This is well suited to purposes such as marking cell lineages, but for monitoring dynamic processes such as gene expression or chromosome transmission, a less stable protein with a more rapid turnover would be more suitable.

The role of controlled protein degradation as a method of regulating many cellular biochemical processes has only recently been fully appreciated and the pathways involved in the accurate targetting of proteins for proteolysis are an increasingly important area of study. Failure of the key components in these pathways can be causative in human disease ( Kishino et al., 1997; Matsuura et al., 1997) and responsible for incomplete cell-cycle progression ( Ghislain et al., 1993; Finley et al., 1994; Pu and Osmani, 1995). Central to this process of degradation is the 26S proteosome, a 2000 kDa multi-protein complex which is distinct from the compartmentalized lysosomal proteolysis machinery and is responsible for the ATP-dependent turnover of 80-90% of the cell's protein content ( Lee and Goldberg, 1998). Proteins are generally, but not exclusively, identified for systematic degradation in the proteosome by ubiquitination. The specificity of this process is in part defined by the action of a family of E2-ubiquitin conjugating enzymes, which catalyse the final ubiquitin addition, but is also conferred by sequence motifs in the target protein which act as proteolytic signals.

One such motif, the PEST sequence, is found extensively in short-lived proteins including metabolic enzymes, transcription factors and their regulators, signalling pathway components and certain cyclins (Rechsteiner and Rogers, 1996). PEST sequences are enriched for proline, glutamate, serine and threonine in a negatively or neutrally charged background and removal of this region from short-lived proteins results in more stable derivatives ( Tyers et al., 1992; Pu and Osmani, 1995; Tsurumi et al., 1995). The metabolic enzyme ornithine decarboxylase (ODC) is a key regulatory control point in polyamine biosynthesis and is regulated by 26S proteosome degradation, albeit without prior ubiquitination, which is mediated by two PEST regions. Transfer of the carboxy-terminal ODC PEST region to a reporter enzyme, dihydrofolate reductase, resulted in an increased turnover rate of the DHFR protein ( Loetscher et al., 1991).

An alternative mechanism of targetting proteins for rapid degradation is the `cyclin destruction box' (CDB), which leads to ubiquitination and degradation of A- and B-type cyclins by the 26S proteosome at the end of mitosis. This process is promoted by the anaphase promoting complex (APC), which possesses E3-ubiquitin ligase activity, as part of its role as regulator of the metaphase-anaphase transition checkpoint. Transfer of the CDB to protein A led to destabilization of this protein in Xenopus extracts ( Glotzer et al., 1991).

We wish to use destabilized GFPs to assay chromosome transmission in mammalian cells. We therefore engineered fusion proteins that link GFP to an ODC PEST sequence and a cyclin B1 destruction box region, to determine if these signals can reduce the resistance of wild-type GFP to proteolysis. The stabilities of these fusion proteins in mouse cells were compared with that of unmodified GFP.

Materials and methods

Plasmid construction

All constructs were produced in a modified version of the high-level mammalian constitutive expression vector pCAGG-Zeo ( Niwa et al. 1991), allowing coordinated expression of GFP and Zeocin (Invitrogen) drug resistance from a single transcript incorporating an internal ribosome entry site. The `humanized' S65T variant of GFP ( Heim et al. 1995) was cloned as a 720 bp Ecl136II/ XbaI fragment into EcoRI-cut pCAGG-Zeo, after end-filling of recessed 3′ ends. In the resulting plasmid, the GFP gene was flanked by reconstituted Eco RI sites, the downstream 3′ site was inactivated to facilitate subsequent cloning steps. The plasmid was further modified by removal of a 760 bp region containing a polyoma virus origin of replication resulting in the 7.8 kb plasmid pCAGG-GFP.

The carboxy-terminal PEST sequence corresponding to codons 423-449 of the mouse ornithine decarboxylase gene (mODC) was amplified by PCR from LA-9 genomic DNA using the primers ODC-1 (5′-GAG CTG TAC AAG CAT GGC TTC CCG CCG GAG-3′) and ODC-2 (5′-GAG CTG TAC A TT AAC GGT CCA TCC CGC TCT C-3′) which contain BsrGI sites (underlined). A 100 bp product was isolated and cloned into the unique Bsr GI site at the 3′ end of the GFP gene in pCAGG-GFP to produce an in-frame fusion of the PEST sequence to the carboxy terminal of the GFP protein. Sequence analysis of this plasmid, pGFP-PEST revealed a single base deletion in the PCR-derived fragment at the intended TAA termination codon (in bold). This frameshift mutation resulted in the addition of a sequence of nine amino acids (NVQVKLPRL), not found in either mODC or GFP, before a TAG termination codon was reached (Figure 1A).

Cell-cycle specific degradation of GFP was achieved using an amino-terminal fusion of the mouse cyclin B1 destruction box (CDB) motif to GFP. A 348 bp fragment from the 5′ end of mouse cyclin B1 was amplified by PCR from embryonic stem cell genomic DNA using primers CYC-1 (5′-CGG AAT TCT CTG ATT TTG GAG GAG CCA TG-3′) and CYC-2 (5′-ATG AAG CTT TTC AAG TTC AGG TTC AGG C-3′) which included EcoRI and HindIII restriction sites (underlined), respectively. The resulting amplified product was cloned, after digestion, into partial HindIII/ EcoRI-cut pCAGG-GFP producing an in-frame fusion to the N-terminal end of GFP, resulting in plasmid pCDB-GFP. Five amino acids (KLAAT), derived from vector sequences, separate the 3′ end of the cyclin fragment from the original GFP initiation codon. Sequencing confirmed the cyclin sequence was free of PCR-induced mutations and corresponded to a published variant murine B1 cyclin ( Hanley-Hyde et al., 1992).

Sub-cloning of the mODC PEST sequence from pGFP-PEST into the BsrGI site of pCDB-GFP resulted in plasmid pCDB-GFP-PEST in which both putative proteolytic targetting signals were incorporated in the same GFP fusion protein.

Cell culture and transfections

Mouse cell line LA-9 was maintained in DMEM supplemented with 10% fetal calf serum (Globepharm) and selection for drug-resistant lines made with 600 μg/ml Zeocin. Cell lines with stable integrations of the various GFP constructs were produced by electroporation of LA-9 cells with 10 μg of linearized plasmid DNA. Colonies were visualized and selected for GFP expression using an inverted fluorescence microscope (Leica DMIRB) with an FITC filter set (Leica I3) and several independent colonies chosen for expansion and subsequent analysis. For analysis of the CDB-GFP dynamics, cells were synchronized in S phase by treatment with 2 μg/ml aphidicolin (Sigma) for 16 h. FACS analysis of GFP fluorescence was performed on a Becton-Dickinson Facscalibur machine using the supplied FITC filter set.

Metabolic labelling of proteins and immunoprecipitation

GFP half-life was determined by immunoprecipitation of pulse-labelled proteins. Approximately 1×10 ⁶ cells (30-40% confluent in 10 cm plates) were incubated with 50 μCi of ³⁵S-labelled methionine (TranSLabel, ICN Pharmaceuticals) in 4 ml of methionine-deficient DMEM (Sigma) for 4 h at 37°C to pulse label all proteins including GFP. After this time, the medium was replaced with fully supplemented, unlabelled DMEM and an initial sample taken to determine the incorporation of the ³⁵ S-labelled methionine at the start of the experiment. Between five and seven further samples were taken during the next 48 h chase period at approximately 6 h time points.

For each time point, cells were lysed in 1 ml of TETN250 buffer (25 mM Tris-Cl pH 7.5, 5 mM EDTA, 1% Triton X-100, 250 mM NaCl, 1 mM PMSF), the soluble fraction pre-cleared with rabbit serum and GFP selectively immunoprecipitated using 1 μl of undiluted rabbit anti-GFP polyclonal antibody (IgG fraction; Clontech). GFP-antibody complexes were collected using a 10% formalin-fixed Staphylococcus aureus cell suspension (Immunoprecipitin; Life Technologies) and washed with lysis buffer before separation on 12% SDS-PAGE gels by standard methods ( Harlow and Lane, 1988). Gels were dried under vacuum after fixing in methanol-acetic acid-glycerol and electronic autoradiography was carried out using an Instant Imager (Packard Bell Instruments). The total radioactivity in bands corresponding to GFP or its tagged variants was calculated by volume integration using the built-in software after background correction. The GFP half-life was calculated by linear regression analysis of log(total radioactivity per band) against time.

Results

Stably transformed cell lines containing each of the three fusion protein constructs or the S65T unmodified CAGG- GFP (Figure 1A) were produced in mouse LA-9 cells. The integrity of the fusion proteins produced was determined both by fluorescence microscopy and by immunoprecipitation. Visually, all cell lines displayed green fluorescence, which indicated that they were expressing the GFP fusions although with intensity levels and distribution patterns specific for each construct (described below). Immunoprecipitation of ³⁵S-labelled proteins using a polyclonal anti-GFP antibody produced bands at the predicted size for each of the fusion proteins, from GFP at 27 kDa ( Tsien, 1998) to the combination-tagged CDB-GFP-PEST variant at 43 kDa (Figure 1B). Several additional bands, 39 kDa or larger, were also observed even in the parental untransfected LA-9 control lane and represent non-GFP proteins with affinity for the polyclonal antibody. While one of these bands co-migrates with the CDB-GFP fusion protein, the relative intensity of the bands indicates that this was an unrelated band and did not result from contamination of the parental cell line, which was consistently non-fluorescent.

Immunoprecipitated samples of pulse-chase labelled protein extracts were taken over a 48 h period to determine the stabilities of each of the fusion proteins (Figure 2). As predicted from the structure, CAGG-GFP was a stable protein whose destruction followed first-order kinetics with a half-life of approximately 26 h. The fusion protein containing the C-terminal mouse ODC PEST sequence was also degraded with first-order kinetics, but at an increased rate, resulting in a reduced half-life of 9.8 h. This represents a significant destabilization of the protein of 2.6-fold compared to CAGG-GFP.

There was no significant difference in fluorescence intensities between GFP and GFP-PEST lines when examined visually despite the increased turnover of the fusion protein. This observation was quantitatively confirmed by subsequent FACS analysis and indicates that the steady-state levels of the protein are the same. This was not the case for CDB fusion proteins that showed highly variable levels of expression between individual cells from unsynchronized cultures of the same clonal line. In particular, cells undergoing mitosis (as determined by a `rounding off' phenotype) or cells which had recently completed cytokinesis appeared non-fluorescent and revealed a bimodal distribution in FACS analysis (Figure 3D). The left-hand peak in this profile corresponded to cells undergoing mitosis which, nevertheless, showed minimal levels of fluorescence 2-3 times brighter than the non-fluorescent parental LA-9 cells (Figure 3B). During this period of the cell cycle, many proteins are being degraded by the 26S proteosome and so complete removal of the excessive amounts of GFP produced may be hindered by saturation of the degradation machinery.

The cyclin destruction box produced the most significant effect on protein stability, as the CDB-GFP fusion protein had a half-life of 5.8 h averaged over the 48 h sample period (Figure 2). However, this rate was not constant during this time, as demonstrated when synchronized, rather than unsynchronized, cultures were examined (Figure 3A). In this case, the majority of GFP turnover was seen to occur as cells entered mitosis, during which the GFP half-life was 4.9 h. Outside the mitotic phase, the protein remained predominantly stable. Thus, the figure of 5.8 h reflects the fact that not all cells in the unsynchronized culture are actively degrading GFP to the same extent.

The addition of the PEST motif to this CDB-GFP protein marginally reduced the average half-life further to 5.5 h. The outstanding feature of this latter `combination' fusion is that overall levels of fluorescence were the lowest for any of the fusions, such that no labelled protein could be detected on the final time point sample. Also, FACS analysis of unsynchronized cells expressing CDB-GFP-PEST showed a bimodal distribution of fluorescence where the lower peak coincided with the non-fluorescent negative control (data not shown). Thus, addition of the PEST region does not appear to make a significant difference to fluorescence when protein levels are non-limiting; at lower concentrations, its effect is more evident.

The most prominent consequence of fusing the GFP sequence to the cyclin destruction box was highlighted through a time-dependent analysis of fluorescence development and decay in cells from synchronized culture (Figure 3A and F). Cells from lines containing either the CAGG-GFP or CDB-GFP variants were blocked in S phase using the DNA synthesis inhibitor aphidicolin ( Sourlingas and Sekeri-Pataryas, 1996). The FACS profiles of these cells immediately after removal of the synthesis block showed similar distributions of fluorescence intensities, with wild-type GFP being slightly higher (Figure 3C and E). However, over a period of 26 h, corresponding to approximately one and a half cell cycles for LA-9 cells, the population profiles differed dramatically. In order to measure the early stages of fluorescence loss, observed as an initial moderate shift in the FACS profile peak to the left in Figure 3D, cells were defined as fluorescent if they exhibited a relative fluorescence intensity of ≥200 units. Inevitably, below this threshold some cells were included as non-fluorescent despite expressing significant levels of GFP. By this criterion, the percentage of fluorescent cells remained constant (80-85%) in the line expressing CAGG-GFP, whereas the CDB-GFP line showed a loss of fluorescence down to 30% as the synchronized cells entered mitosis. Fluorescence levels increased subsequently as cells progressed through G1 and S phase before a second decrease occurred as cells entered their second mitotic division. The reduced peak in fluorescence levels during this second cycle (62% compared with 78%) results from the loss of synchronization in the population after this length of time. These dynamic changes in CDB-GFP fluorescence with cell cycle progression are illustrated in Figure 3F, in which each cell of a synchronized pair undergoes mitosis resulting in reductions in fluorescence levels. At the four-cell stage, once the cells are in interphase, fluorescence levels have returned to a maximum and are comparable between all cells. Although it is not possible from these data to establish the exact timing of these changes with respect to the cell cycle, the pattern is consistent with that reported for normal cyclin B1 accumulation and degradation ( Glotzer et al., 1991).

Discussion

The most notable impact of green fluorescent protein technology to date in cell biology has been its use in fusion proteins for monitoring a specific protein's localization, trafficking and processing ( Corbett et al., 1995; Rizzuto et al., 1996; Oatey et al., 1997; Tsien, 1998). For these purposes, GFP already has been engineered to be brighter and to exhibit a wider range of excitation and extinction frequencies, which are amenable to double labelling studies ( Tsien, 1998). However, further development of GFP will undoubtedly take the direction of utilizing GFP per se as an indicator of cellular state, as opposed merely to conferring fluorescence on previously non-fluorescent proteins of interest. It is to this class of modification that the GFP variants described in this work belong.

The reduction in GFP stability seen in the mODC PEST-tagged variant, while significant, still places GFP at the higher end of stability compared with more traditional reporters of gene activity such as luciferase which has a half-life of approximately 3 h ( Thompson et al., 1991). In addition, the lack of enzymatic amplification of a signal with GFP means that its sensitivity is limited, as cytoplasmic concentrations of approximately 1 μM are required to distinguish signal from auto-fluorescent background ( Niswender et al., 1995). However, as most enzymatic methods involve cell disruption, this improvement in using GFP non-invasively with a shorter half-life will be suitable for many applications. In particular, for monitoring chromosome transmission, where expression of GFP to readily detectable levels is not problematic, the shorter half-life means that parentally derived cytoplasmic GFP is removed quickly, allowing the measurement of de novo GFP synthesis from the daughter cells.

GFP may yet be a viable alternative for in vivo gene expression studies if the destabilization can be improved to reduce the half-life further. A destabilized GFP variant, d2GFP, has recently been described ( Li et al., 1998) which utilizes a PEST region very similar to that used in this work. However, the reported half-life of this variant is 2 h and further reports of a 1 h variant (d1GFP) are based on fluorescence measurements, not biochemical purification. The discrepancy between those claims and the results presented here has been investigated to distinguish between cell differences, experimental protocols and intrinsic properties of the GFP variants used. We have confirmed the increased rate of degradation of the d1GFP variant, compared with the GFP-PEST described here (data not shown), and using the more accurate method of pulse-chase labelling we have determined its half-life to be 50 min in human HT1080 cells.

This result is remarkable considering the overall homology between the two mODC PEST sequences used. The d2GFP variant is extended compared with GFP-PEST by only 13 amino acids, but the core PEST region is maintained between both. This clearly implicates `non-PEST motif' residues as having a major contributory role to the rapid turnover of the mouse ornithine decarboxlase protein ordinarily. What is more significant, however, is that in the most unstable d1GFP variant the conserved Glu residues in the PEST region are mutated to Ala. While these changes would theoretically predict a weaker PEST motif, in practice the alterations result in a 50% reduction in protein stability. Consequently, the presence of a PEST motif within a protein merely implies a propensity to instability and cannot indicate the magnitude of the degradation rate even when close to or identical with the consensus PEST sequence.

While the patterns of protein degradation for the cyclin-GFP fusion proteins are more complex than the simple linear profiles obtained with the PEST fusions, these variants are potentially useful as non-invasive indicators of cell-cycle status. The FACS profile of unsynchronized cells expressing these proteins (Figure 3D) is an indirect measurement of the proportion of cells in defined stages of the cell cycle. Although more detailed investigation of the correlation between fluorescence levels and cellular status is necessary, the lower peak of the fluorescence distribution corresponded broadly to mitotic and early G1 cells. Therefore, this form of GFP could be used as an alternative to procedures, such as propidium iodide staining, which require fixation with concomitant cell death. Indeed, in the cell synchronization experiments described (Figure 3A-E), the fluorescence levels were a direct indication of the efficiency of the aphidicolin treatment.

However, it is still debatable as to how accurately GFP fluorescence intensity is a reliable indicator of GFP levels in a cell. Two factors that significantly affect the fluorescent properties of GFP, namely the requirement for post-translational oxidative fluorophore formation and the sensitivity to cellular pH, could result in fluorescence intensities being lower than actual GFP concentration. Western analysis indicates that a good correlation exists between fluorescence intensity and protein concentration (Li et al., 1998), but does not distinguish between immature, non-fluorescent and mature, fluorescent forms of GFP.

Considering this correlation between fluorescence levels and protein content, it is paradoxical that cell lines containing the unstable GFP-PEST construct (half-life 9.8 h) have fluorescence intensities not significantly different to those of lines containing its more stable GFP counterpart (half-life 26 h). Differences in steady-state protein levels between these lines would predict that the more unstable lines are also the least intense for GFP fluorescence, contrary to the actual observation. A similar observation was reported for the d2GFP variant ( Li et al., 1998), although in our experimental system lines containing this construct evidently displayed weaker fluorescence both in FACS analysis and by microscopy. Although this could indicate that non-fluorescent immature GFP molecules are preferentially degraded, we believe this to be unlikely. Instead, it is probable that proteolytic products from 26S proteosome activity maintain a degree of fluorescence ( Tsien, 1998) but are not detected by the immunoblot technique used to measure half-life, thereby increasing the apparent GFP concentration. Thus, reduction in fluorescence intensity is proportional to a decrease in GFP half-life, but not to the same magnitude. The d1GFP variant that is 30 times more unstable than the wild-type GFP is, nonetheless, only ~70-80% less intense by FACS analysis.

The results also shed some light on the biology of proteolytic signalling itself. The GFP-PEST fusion construct has been tested in human HT1080 and HeLa-S3 cell lines where the fusion protein localized to give a punctate pattern. The distribution of GFP foci appeared limited to the cytoplasm, partially concentrated around the nuclear envelope (data not shown). This phenotype was only seen in LA-9 cells when the brighter EGFP variant was fused to mouse ODC PEST, although the phenotype was less pronounced than in human cells. This mottled pattern is unlikely to be due to sub-cellular localization of the 26S proteosome, which is known to be found in both the nucleus and cytoplasm (Coux et al., 1996), but may represent natural sequestering of proteins marked for degradation away from the active cellular protein conterparts. Equally, the phenotype may be artefactual, with the very high levels of expression from the pCAGG vector resulting in protein aggregation complexes.

The distribution of the CDB-GFP protein was also consistent with known cyclin B1 characteristics ( Hagting et al., 1998). A pronounced nuclear localization was observed in mouse and human HT1080 cells (Figure 3F, 0 h, lower panel). Nuclear-cytoplasmic translocation is a normal feature of cyclin B1 expression and is mediated by a putative cytoplasmic retention or nuclear export signal between residues 129 and 157 ( Hagting et al., 1998; Toyoshima et al., 1998) which is absent from the CDB-GFP construct (Figure 1A). In this respect, CDB-GFP is a useful starting point for the in vivo analysis of the 5′ region of the gene which is responsible for the dynamic movement of the cyclin B1 protein throughout the cell cycle.

In conclusion, it is evident from the results using the PEST and CDB fusions that the barrel-like structure of GFP is not refractory to major modifications that alter the basic properties of GFP biology. In these examples, GFP stability can be permanently compromised by the addition of the appropriate proteolytic signal sequences. Engineering opportunities are, however, limited to the carboxy- and amino-terminal ends for GFP, as internal modifications destroy the protective barrel structure and expose the fluorophore rendering it non-fluorescent (Dopf and Horiagon, 1996). However, the ease by which new derivatives can be produced and tested directly in living cells will allow GFP to be tailored to a variety of individual uses.

We thank Elizabeth Burns for plasmid donations and Steve Kain and Jason Li of Clontech for sharing unpublished results. P.C. was supported by the MRC and C.T.-S. by the Cancer Research Campaign.

References

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Attenuation of green fluorescent protein half-life in mammalian cells | Protein Engineering, Design and Selection | Oxford Academic

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The half-life of the green fluorescent protein (GFP) was determined biochemically in cultured mouse LA-9 cells. The wild-type protein was found to be stable with a half-life of ~26 h, but could be destabilized by the addition of putative proteolytic signal sequences derived from proteins with shorter half-lives.

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techniques used to monitor the bacteria or virus after being marked

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Learning Objectives

Describe the benefits of immunofluorescent antibody assays in comparison to nonfluorescent assays

Compare direct and indirect fluorescent antibody assays

Explain how a flow cytometer can be used to quantify specific subsets of cells present in a complex mixture of cell types

Explain how a fluorescence-activated cell sorter can be used to separate unique types of cells

Rapid visualization of bacteria from a clinical sample such as a throat swab or sputum can be achieved through fluorescent antibody (FA) techniques that attach a fluorescent marker ( fluorogen) to the constant region of an antibody, resulting in a reporter molecule that is quick to use, easy to see or measure, and able to bind to target markers with high specificity. We can also label cells, allowing us to precisely quantify particular subsets of cells or even purify these subsets for further research.

As with the enzyme assays, FA methods may be direct, in which a labeled mAb binds an antigen, or indirect, in which secondary polyclonal antibodies bind patient antibodies that react to a prepared antigen. Applications of these two methods were demonstrated in Figure 8 in Instruments of Microscopy. FA methods are also used in automated cell counting and sorting systems to enumerate or segregate labeled subpopulations of cells in a sample.

Direct Fluorescent Antibody Techniques

Direct fluorescent antibody (DFA) tests use a fluorescently labeled mAb to bind and illuminate a target antigen. DFA tests are particularly useful for the rapid diagnosis of bacterial diseases. For example, fluorescence-labeled antibodies against Streptococcus pyogenes ( group A strep) can be used to obtain a diagnosis of strep throat from a throat swab. The diagnosis is ready in a matter of minutes, and the patient can be started on antibiotics before even leaving the clinic. DFA techniques may also be used to diagnose pneumonia caused by Mycoplasma pneumoniae or Legionella pneumophila from sputum samples (Figure 1). The fluorescent antibodies bind to the bacteria on a microscope slide, allowing ready detection of the bacteria using a fluorescence microscope. Thus, the DFA technique is valuable for visualizing certain bacteria that are difficult to isolate or culture from patient samples.

Think about It

In a direct fluorescent antibody test, what does the fluorescent antibody bind to?

Indirect Fluorescent Antibody Techniques

Indirect fluorescent antibody (IFA) tests (Figure 2) are used to look for antibodies in patient serum. For example, an IFA test for the diagnosis of syphilis uses T. pallidum cells isolated from a lab animal (the bacteria cannot be grown on lab media) and a smear prepared on a glass slide. Patient serum is spread over the smear and anti-treponemal antibodies, if present, are allowed to bind. The serum is washed off and a secondary antibody added. The secondary antibody is an antihuman immunoglobulin conjugated to a fluorogen. On examination, the T. pallidum bacteria will only be visible if they have been bound by the antibodies from the patient's serum.

The IFA test for syphilis provides an important complement to the VDRL test discussed in Detecting Antigen-Antibody Complexes. The VDRL is more likely to generate false-positive reactions than the IFA test; however, the VDRL is a better test for determining whether an infection is currently active.

IFA tests are also useful for the diagnosis of autoimmune diseases. For example, systemic lupus erythematosus (SLE) (see Autoimmune Disorders) is characterized by elevated expression levels of antinuclear antibodies (ANA). These autoantibodies can be expressed against a variety of DNA-binding proteins and even against DNA itself. Because autoimmunity is often difficult to diagnose, especially early in disease progression, testing for ANA can be a valuable clue in making a diagnosis and starting appropriate treatment.

The IFA for ANA begins by fixing cells grown in culture to a glass slide and making them permeable to antibody. The slides are then incubated with serial dilutions of serum from the patient. After incubation, the slide is washed to remove unbound proteins, and the fluorescent antibody (antihuman IgG conjugated to a fluorogen) added. After an incubation and wash, the cells can be examined for fluorescence evident around the nucleus (Figure 3). The titer of ANA in the serum is determined by the highest dilution showing fluorescence. Because many healthy people express ANA, the American College of Rheumatology recommends that the titer must be at least 1:40 in the presence of symptoms involving two or more organ systems to be considered indicative of SLE.

Think about It

In an indirect fluorescent antibody test, what does the fluorescent antibody bind to?

What is the ANA test looking for?

Flow Cytometry

Fluorescently labeled antibodies can be used to quantify cells of a specific type in a complex mixture using flow cytometry (Figure 4), an automated, cell-counting system that detects fluorescing cells as they pass through a narrow tube one cell at a time. For example, in HIV infections, it is important to know the level of CD4 T cells in the patient's blood; if the numbers fall below 500 per μL of blood, the patient becomes more likely to acquire opportunistic infections; below 200 per μL, the patient can no longer mount a useful adaptive immune response at all. The analysis begins by incubating a mixed-cell population (e.g., white blood cells from a donor) with a fluorescently labeled mAb specific for a subpopulation of cells (e.g., anti-CD4). Some experiments look at two cell markers simultaneously by adding a different fluorogen to the appropriate mAb. The cells are then introduced to the flow cytometer through a narrow capillary that forces the cells to pass in single file. A laser is used to activate the fluorogen. The fluorescent light radiates out in all directions, so the fluorescence detector can be positioned at an angle from the incident laser light.

Figure 4 shows the obscuration bar in front of the forward-scatter detector that prevents laser light from hitting the detector. As a cell passes through the laser bar, the forward-scatter detector detects light scattered around the obscuration bar. The scattered light is transformed into a voltage pulse, and the cytometer counts a cell. The fluorescence from a labeled cell is detected by the side-scatter detectors. The light passes through various dichroic mirrors such that the light emitted from the fluorophore is received by the correct detector.

Data are collected from both the forward- and side-scatter detectors. One way these data can be presented is in the form of a histogram. The forward scatter is placed on the y-axis (to represent the number of cells), and the side scatter is placed on the x-axis (to represent the fluoresence of each cell). The scaling for the x-axis is logarithmic, so fluorescence intensity increases by a factor of 10 with each unit increase along the axis.

Flow cytometry data are often compiled as a histogram. In the histogram, the area under each peak is proportional to the number of cells in each population. The x-axis is the relative fluorescence expressed by the cells (on a log scale), and the y-axis represents the number of cells at a particular level of fluorescence. Figure 5 depicts an example in which a culture of cells is combined with an antibody attached to a fluorophore to detect CD8 cells and then analyzed by flow cytometry. The histogram has two peaks. The peak on the left has lower fluorescence readings, representing the subset of the cell population (approximately 30 cells) that does not fluoresce; hence, they are not bound by antibody and therefore do not express CD8. The peak on the right has higher fluorescence readings, representing the subset of the cell population (approximately 100 cells) that show fluorescence; hence, they are bound by the antibody and therefore do express CD8.

Think about It

What is the purpose of the laser in a flow cytometer?

In the output from a flow cytometer, the area under the histogram is equivalent to what?

Clinical Focus: HIV, Resolution

This example concludes the story that started in Polyclonal and Monoclonal Antibody Production and EIAs and ELISAs.

After notifying all 1300 patients, the hospital begins scheduling HIV screening. Appointments were scheduled a minimum of 3 weeks after the patient's last hospital visit to minimize the risk of false negatives. Because some false positives were anticipated, the public health physician set up a counseling protocol for any patient whose indirect ELISA came back positive.

Of the 1300 patients, eight tested positive using the ELISA. Five of these tests were invalidated by negative western blot tests, but one western blot came back positive, confirming that the patient had indeed contracted HIV. The two remaining western blots came back indeterminate. These individuals had to submit to a third test, a PCR, to confirm the presence or absence of HIV sequences. Luckily, both patients tested negative.

As for the lone patient confirmed to have HIV, the tests cannot prove or disprove any connection to the syringes compromised by the former hospital employee. Even so, the hospital's insurance will fully cover the patient's treatment, which began immediately.

Although we now have drugs that are typically effective at controlling the progression of HIV and AIDS, there is still no cure. If left untreated, or if the drug regimen fails, the patient will experience a gradual decline in the number of CD4 helper T cells, resulting in severe impairment of all adaptive immune functions. Even moderate declines of helper T cell numbers can result in immunodeficiency, leaving the patient susceptible to opportunistic infections. To monitor the status of the patient's helper T cells, the hospital will use flow cytometry. This sensitive test allows physicians to precisely determine the number of helper T cells so they can adjust treatment if the number falls below 500 cells/µL.

Cell Sorting Using Immunofluorescence

The flow cytometer and immunofluorescence can also be modified to sort cells from a single sample into purified subpopulations of cells for research purposes. This modification of the flow cytometer is called a fluorescence-activated cell sorter (FACS). In a FACS, fluorescence by a cell induces the device to put a charge on a droplet of the transporting fluid containing that cell. The charge is specific to the wavelength of the fluorescent light, which allows for differential sorting by those different charges. The sorting is accomplished by an electrostatic deflector that moves the charged droplet containing the cell into one collecting vessel or another. The process results in highly purified subpopulations of cells.

One limitation of a FACS is that it only works on isolated cells. Thus, the method would work in sorting white blood cells, since they exist as isolated cells. But for cells in a tissue, flow cytometry can only be applied if we can excise the tissue and separate it into single cells (using proteases to cleave cell-cell adhesion molecules) without disrupting cell integrity. This method may be used on tumors, but more often, immunohistochemistry and immunocytochemistry are used to study cells in tissues.

Watch this videos to learn more flow cytometry:
This site shows how a FACS works.
Think about It

In fluorescence activated cell sorting, what characteristic of the target cells allows them to be separated?

Table 1 compares the mechanisms of the fluorescent antibody techniques discussed in this section.

Table 1. Fluorescent Antibody Techniques

Direct fluorescent antibody (DFA)

Uses fluorogen-antibody conjugates to label bacteria from patient samples

Visualizing Legionella pneumophila from a throat swab

Indirect fluorescent antibody (IFA)

Detects disease-specific antibodies in patent serum

Diagnosing syphilis; detecting antinuclear antibodies (ANA) for lupus and other autoimmune diseases

Flow cytometry

Labels cell membranes with fluorogen-antibody conjugate markers excited by a laser; machine counts the cell and records the relative fluorescence

Counting the number of fluorescently labeled CD4 or CD8 cells in a sample

Fluorescence activated cell sorter (FACS)

Form of flow cytometry that both counts cells and physically separates them into pools of high and low fluorescence cells

Sorting cancer cells

Key Concepts and Summary

Immunofluorescence assays use antibody-fluorogen conjugates to illuminate antigens for easy, rapid detection.

Direct immunofluorescence can be used to detect the presence of bacteria in clinical samples such as sputum.

Indirect immunofluorescence detects the presence of antigen-specific antibodies in patient sera. The fluorescent antibody binds to the antigen-specific antibody rather than the antigen.

The use of indirect immunofluorescence assays to detect antinuclear antibodies is an important tool in the diagnosis of several autoimmune diseases.

Flow cytometry uses fluorescent mAbs against cell-membrane proteins to quantify specific subsets of cells in complex mixtures.

Fluorescence-activated cell sorters are an extension of flow cytometry in which fluorescence intensity is used to physically separate cells into high and low fluorescence populations.

Multiple Choice

Suppose you need to quantify the level of CD8 T cells in the blood of a patient recovering from influenza. You treat a sample of the patient's white blood cells using a fluorescent mAb against CD8, pass the cells through a flow cytometer, and produce the histogram shown below. The area under the peak to the left (blue) is three times greater than the area of the peak on the right (red). What can you determine from these data?

1. There are no detectable CD8 cells.

2. There are three times as many CD4 cells than CD8 cells.

3. There are three times as many CD8 cells than CD4 cells.

4. CD8 cells make up about one-fourth of the total number of cells.

In the data described in the previous question, the average fluorescence intensity of cells in the second (red) peak is about ________ that in the first (blue) peak.

In a direct fluorescent antibody test, which of the following would we most likely be looking for using a fluorescently-labeled mAb?

1. bacteria in a patient sample

2. bacteria isolated from a patient and grown on agar plates

3. antiserum from a patient smeared onto a glass slide

4. antiserum from a patient that had bound to antigen-coated beads

Fill in the Blank

In flow cytometry, cell subsets are labeled using a fluorescent antibody to a membrane protein. The fluorogen is activated by a(n) ________ as the cells pass by the detectors.

Fluorescence in a flow cytometer is measured by a detector set at an angle to the light source. There is also an in-line detector that can detect cell clumps or ________.

Think about It

1. A patient suspected of having syphilis is tested using both the VDRL test and IFA. The IFA test comes back positive, but the VDRL test is negative. What is the most likely reason for these results?

2. A clinician suspects that a patient with pneumonia may be infected by Legionella pneumophila. Briefly describe two reasons why a DFA test might be better for detecting this pathogen than standard bacteriology techniques.

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Describe the benefits of immunofluorescent antibody assays in comparison to nonfluorescent assays Compare direct and indirect fluorescent antibody assays Explain how a flow cytometer can be used to quantify specific subsets of cells present in a complex mixture of cell types Explain how a fluorescence-activated cell sorter can be used

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Fluorescence microscopy for disease diagnosis and environmental monitoring 

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Fluorescence microscopy for disease diagnosis and environmental monitoring 

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Background on fluorescent marking of antibodies and how it is done.

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The lupus band test in systemic lupus erythematosus patients

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The lupus band test in systemic lupus erythematosus patients

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Example of a specific skin diseases Lupus that uses fluorescent labelled antibodies for diagnostics

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Abstract

The lupus band test (LBT) is a diagnostic procedure that is used to detect deposits of immunoglobulins and complement components along the dermoepidermal junction in patients with lupus erythematosus (LE). The LBT is positive in about 70%-80% of sun-exposed non-lesional skin specimens obtained from patients with systemic LE (SLE), and in about 55% of SLE cases if sun-protected nonlesional skin is analyzed. In patients with cutaneous LE only, the lesional skin usually shows a positive LBT. The LBT helps in differentiating LE from other similar skin conditions and may also be helpful in making the diagnosis of SLE in subjects with no specific cutaneous lesions. Furthermore, a positive LBT may be applied as a prognostic parameter for LE patients. However, the correct interpretation of this test requires detailed knowledge of the site of the biopsy, deposit components, morphology and brightness of the immunofluorescent band, and other associated serologic findings, as well as the response to treatment. It must be emphasized that LBT is a laboratory procedure that should always be interpreted in conjunction with clinical findings and other serological and immunopathological parameters.

Keywords: lupus erythematosus, dermoepidermal junction, diagnostics

Introduction

Lupus erythematosus (LE) is an autoimmune disease affecting different body systems, including, but not limited to, joints, skin, kidneys, blood, heart, and brain, and may be characterized by the production of various autoantibodies, complement consumption, and the presence of circulating immune complexes. 1 As in other autoimmune diseases, the autoimmune reaction in LE, driven by various components of the immune system, results in inflammation and tissue damage. Systemic LE (SLE), cutaneous LE (CLE) (including subacute CLE [SCLE] and discoid LE [DLE]), drug-induced LE, and neonatal LE are the major subtypes of LE. Of these, SLE is the most serious form. It occurs about 10 times more often among women than among men, usually in young adults, and Black and Asian people seem to be more commonly affected. 2, 3 This is a life-threatening disease, sometimes with a fatal outcome. Fortunately, the medical improvement of patients' care in the past few decades makes treatment failure increasingly rare. Currently, the 10-year survival rate in developed countries is estimated to be above 90%. 4, 5

The histology of the diseased skin of LE patients shows some typical characteristics, including vacuolar degeneration of the basal layer with thickened subepidermal basement membrane, as well as perivascular and periadnexal lymphocytic infiltrates, which may help to establish the diagnosis. 1, 6 In addition, lesional and nonlesional skin may show deposits of immunoglobulins along the dermoepidermal junction (DEJ), which is a unique feature of LE. 6

Definition of lupus band test (LBT)

Deposits of immunoglobulins at the DEJ, together with the thickened basement membrane, in lesional skin of LE patients were first described by Burnham et al. 7 Subsequently, Cormane 8 demonstrated similar deposits in clinically normal skin of SLE, but not in that of CLE subjects. Detection of these deposits of immunoglobulins and complement components in the skin of patients with LE, demonstrable as a linear band at the basement membrane zone, was then named the lupus band test (LBT). All major immunoglobulin classes (IgG, IgM, and IgA) and various complement components have been identified in these DEJ deposits. 6 The test is done on the skin biopsy, usually with direct immunofluorescence staining, but immunohistochemistry may also be applied. Importantly, LBT can be helpful in distinguishing SLE from CLE, because in SLE patients the LBT is frequently positive in both involved and uninvolved skin, whereas in CLE patients only the involved skin is positive.

Although the exact mechanism of the immunoglobulin deposition at DEJ in LE patients is not clear, it is believed that these immunoglobulins are not antibodies against basement membrane zone components but rather represent, at least partly, circulating immune complexes of DNA and antinuclear antibodies trapped within the DEJ. Furthermore, DNA released from ultraviolet-injured keratinocytes, although diffusing across the basement membrane zone, may bind to collagen IV and then serve as an antigen for circulating antinuclear antibodies. 6, 9

Sensitivity and specificity of the LBT

The sensitivity and specificity of the LBT are strictly related to the body area tested and the criteria used for assigning the test as positive. For instance, in the study by Cardinali et al 10 on the sun-protected nonlesional skin of SLE patients, the sensitivity of the LBT varied between 10.5% and 78.9% and the specificity between 47.8% and 97.8%, depending on the criteria used by the authors. The most frequent immunoglobulin class deposited is IgM, which is seen in about 90% of lesional skin biopsies, whereas the least frequently seen class is IgA. 6, 11 However, a weak decoration with IgM along the DEJ is also common in sun-exposed skin of patients who do not have LE. It was demonstrated that healthy sun-exposed skin may show a weak interrupted linear or granular pattern of IgM and C1q deposits at the DEJ in nearly 25% of samples, but only 5% of them showed the presence of IgG, IgA, or complement component 3 (C3). 12, 13 Taking these observations into account, in line with Crowson and Magro, 6 we suggest that LBT should be considered positive when deposits of IgM in sun-exposed skin form a continuous band over at least 50% of the width of the biopsy specimen which is at least moderate in intensity. The predictive value of LBT for SLE was greater with C4 (100%), properdin (91.3%), and IgA (86.2%) than with IgM (59%). 6 The specificity and predictive value also increase with the number of immunoreactants detected at the DEJ. 6 In sun-protected skin, an interrupted band of IgM of at least moderate intensity is usually sufficient for designation as a positive LBT. 6 The deposition of IgG is usually less intense than that of IgM, and false-positive results are practically nonexistent. 6

Altogether, the LBT in patients with SLE is positive in about 70%-80% subjects when sun-exposed nonlesional skin is tested, and in about 55% cases when sun-protected nonlesional skin is studied. In patients with CLE, the LBT of nonlesional skin is usually negative, although the lesional skin may frequently show immune deposits at the DEJ. 6 Moreover, a positive LBT may serve as a prognostic indicator in patients with an established diagnosis of LE, as it correlates with severe extracutaneous disease, mainly lupus nephritis, and with anti-dsDNA antibodies. 6, 11, 14, 15

Staining patterns of the LBT

The immunoglobulin staining pattern in nonlesional LE skin at low magnification is usually described as granular or closely spaced vertically oriented fibrils, and sometimes also as a thick, homogeneous band (). The C3 deposits are the most common component, and IgM was the most frequent immunoglobulin class observed in the LBT. 16 Remarkably, a sharply defined, thin linear band at the DEJ seen in the pemphigoid should not be considered as a positive LBT (). 6 Under high power, the patterns of immunoglobulin deposition at the DEJ may be homogenous (), fibrillar, stippled (), shaggy (), lumpy (), linear, or thready. 6, 11 All these patterns are seen in a continuous fashion. A discontinuous or interrupted LBT is less specific and can be seen in a number of other disorders such as actinic keratosis, polymorphic light eruption, and rosacea and in normal sun-exposed skin (). 11 Ultrastructurally, these immunoreactants are deposited on or below lamina densa of the basement membrane. 11

Table 1

Positive lupus band test

Granular band or closely spaced, vertically oriented fibrils at dermoepidermal junction; sometimes a thick, homogeneous band.

Healthy sun-exposed skin

May demonstrate positive lupus band test.

The band is usually less intensive and frequently focal or interrupted.

No reactivity is seen in sun-protected skin.

Autofluorescence of dermal collagen and elastin fibers

Might simulate a positive lesional lupus band test.

Artifactual nature of this false-positive finding becomes apparent at higher magnification.

Bullous pemphigoid

Sharply defined thin linear band at dermoepidermal junction.

Presence of circulating antibodies against basement membrane components is helpful for a correct diagnosis.

Porphyrias

Fluorescence of the dermoepidermal junction is less intense than that found in dermal blood vessels, which is exactly the reverse of what is seen in lupus erythematosus.

Complement is rarely found in the band.

Rosacea

The band is less intensive and frequently focal or interrupted.

Polymorphic light eruption

The band is less intensive and frequently focal or interrupted.

A stippled pattern of the LBT, consisting of multiple small round points of fluorescence (), is the type most commonly seen in clinically normal skin of SLE. In some patients, sparser, more elongated and threadlike stipples can be seen. A homogenous or solid band of well demarcated, bright fluorescence is seen mostly in chronic atrophic or hypertrophic skin lesions. A thready pattern consisting of short, closely set, bright threads or fibrils is seen in more acute erythematous, edematous lesions. Shaggy, fibrillar, lumpy, and granular patterns have also been seen, usually in a continuous pattern along the DEJ. 11

Sometimes, a false-negative reaction can be observed due to high levels of extravascular IgG deposits in the dermis. 11 Moreover, considerable anatomic regional variations may occur in positive lesional LBT with a cephalocaudal gradient, with lesions on the head more often being positive than those on the trunk. 11

Other staining methods in LE

Detection of the C 5b-9 macromolecular structure, which is formed following activation of the classical or alternative component pathway, is another useful tool for the diagnosis of LE. 1 Intense granular deposition of C 5b-9 along the DEJ is seen in about 80% of lesional skin in patients with SLE. 17 Vascular decoration for C 5b-9 was observed in SLE subjects with anti-Ro/SSA antibodies, in patients with circulating lupus anticoagulant, or when vasculitis is demonstrated in the biopsy. 1 Regarding nonlesional skin of SLE patients, C 5b-9 immunoreactivity might be observed within keratinocytes, especially in subjects with positive anti-ENA antibodies (ENA: Ro, La, Sm, U1RNP). In SCLE and DLE, deposition of C 5b-9 along the DEJ was observed in 66% and 60% of cases, respectively. 18 However, in SCLE, keratinocytes may show granular nuclear or cytoplasmic reactivity for C 5b-9, a finding that is usually absent in DLE. 1, 18

Recently, it was also shown that detection of C3d and C4d by immunohistochemistry might be used for diagnosis of LE as well. 18 C4d is a stable component of classical complement activation and represents a degradation product of C4b. 19 C3d is also a stable component of complement activation, although it can be formed in both classical or alternative complement activation. 19 As shown by Magro and Dyrsen, 19 all DLE cases showed granular, moderate to marked intensity C3d deposits at DEJ, whereas C4d deposits were found in only 15% of DLE patients. Interestingly, all SCLE subjects were negative for C3d and C4d staining along the DEJ, although some of them showed deposition of C3d or C4d in blood vessels, a finding that was also noted in DLE. Similarly to DLE, all samples of SLE demonstrated prominent granular C3d deposition along the DEJ. Remarkably, all SLE patients also had C4d deposits at the DEJ. 19

Application of the LBT

The usefulness of the LBT as a diagnostic procedure in LE patients is well established. The positive result of this test within the uninvolved skin is a strong indicator of LE. 11 The sensitivity of the LBT for diagnosing an active disease seems to be even higher than the value of other laboratory parameters, including serum C3 and C4 levels, erythrocyte sedimentation rate, lymphocyte count, or the presence of anti-dsDNA antibodies (the last two parameters are included in the diagnostic criteria of SLE).

The diagnostic specificity of LBT for LE differentiation from other inflammatory cutaneous conditions that are clinically similar, such as polymorphic light eruption or benign lymphocytic infiltrate of Jessner and Kanoff, is also high. 11, 16 Furthermore, a positive LBT in fully sun-protected nonlesional skin from the buttocks or inner aspect of the upper arm consisting of three or more components (IgA, IgG, IgM, or complement proteins) seems to have the highest specificity for SLE than any other test. 11

The LBT may also be helpful in distinguishing SLE from CLE. Various studies have shown that SLE subjects have immunoglobulin and/or complement deposits at the DEJ in both involved and uninvolved skin, whereas CLE patients had only positive LBT in lesional skin. Therefore, it is essential to biopsy the appropriate skin site for each patient in order to avoid false-negative results. Furthermore, the LBT can be helpful in making a diagnosis of SLE in patients without cutaneous lesions. A positive LBT in clinically normal skin provides early confirmation of SLE, even in patients without LE lesions, where neither a biopsy nor LBT can be performed on a skin lesion. 11 Importantly, test positivity was uninfluenced by steroid and immunosuppressive therapy. 20 The test appears to be of great interest for use on all patients who fail to meet the criteria for the diagnosis of SLE but whose conditions suggest such diagnosis. 20 In addition, the LBT facilitates differentiation of diagnosis of SLE from other antinuclear antibody-positive diseases like rheumatoid arthritis, scleroderma, dermatomyositis, and mixed connective tissue disease. 1, 11 However, occasional positive LBTs were found in lesional skin of dermatomyositis and systemic sclerosis, and this finding has been correlated with a more severe disease course. 18, 21

A positive LBT may also be a predictive value for the prognosis of LE patients. The deposition of IgG in nonlesional sun-protected skin is correlated with anti-dsDNA antibodies titer and with higher incidence of renal disease, although a negative LBT does not necessarily exclude the possibility of renal involvement. 11, 20, 22 A positive LBT within sun-protected normal skin also indicates decreased long-term survival. 11, 22 Recently, Zecević et al, using the SLE Disease Activity Index score, confirmed that the disease was significantly more active in patients with positive LBT on sun-protected nonlesional skin and in those with a higher number of deposited immunoreactants. Moreover, almost all patients with renal involvement had a positive LBT. 23 On the other hand, patients with pure IgM deposition in clinically normal skin have anti-dsDNA antibodies restricted to the IgM class and tend to have a less severe disease course. In addition, C1q deposits along the DEJ in the skin of SLE patients may reflect the presence of DNA at the DEJ, and such patients have a higher index of disease activity. 1, 14, 15

Conclusion

The LBT is a useful diagnostic tool for LE patients. However, the correct interpretation of this test requires detailed knowledge of several correlates, such as the site of the biopsy (lesional or normal skin, sun-protected versus sun-exposed skin), deposit components, morphology and brightness of the immunofluorescent band, and other associated serologic findings, as well as the response to treatment. A positive LBT on sun-protected nonlesional skin represents a sensitive and specific criterion for identifying patients with LE. Furthermore, the LBT on sun-protected normal skin may be helpful in diagnosing SLE in patients with inconclusive clinical and serological profiles and may also be of prognostic significance, particularly if all three (IgG, IgM, and IgA) immunoglobulins are found at the DEJ. Importantly, the LBT is a laboratory procedure that should always be interpreted in conjunction with clinical findings and other serological and immunopathological parameters in order to make a correct diagnosis.

Footnotes
Disclosure
The authors report no conflicts of interest in this work.

References

6. Crowson AN, Magro CM. Cutaneous histopathology of lupus erythematosus. Diagn Histopathol. 2009;15:157-185.

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The lupus band test in systemic lupus erythematosus patients

# data[].attributes.attachment_link.description
The lupus band test (LBT) is a diagnostic procedure that is used to detect deposits of immunoglobulins and complement components along the dermoepidermal junction in patients with lupus erythematosus (LE). The LBT is positive in about 70%-80% of sun-exposed non-lesional skin specimens obtained from patients with systemic LE (SLE), and in about 55% of SLE cases if sun-protected nonlesional skin is analyzed.