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18/10/16: Immunohistochemistry tumor detection 

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18/10/16: Immunohistochemistry tumor detection 

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http://www.urotoday.com/recent-abstracts/urologic-oncology/investigative-urology/91984-overexpression-of-autotaxin-is-associated-with-human-renal-cell-carcinoma-and-bladder-carcinoma-and-their-progression.html

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1/4/16: Immunofluorescence imaging development 

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1/4/16: Immunofluorescence imaging development 

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http://www.news-medical.net/whitepaper/20160401/Analysis-of-High-Specificity-Cellular-Proteins-Using-Multicolor-Immunofluorescence-Imaging.aspx

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28/10/16: Immunofluoresence news article - Activation of two genes linked to development of atherosclerosis

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28/10/16: Immunofluoresence news article - Activation of two genes linked to development of atherosclerosis

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https://www.sciencedaily.com/releases/2016/10/161028113724.htm

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2. Procedure (Pelczar, M and Chan, E. 2010).

1.     Add test sample and labelled antigen to well and incubate

2.     Wash well contents to remove unbound antigen

3.     Add unlabelled antigen to test sample

4.     Radioactivity measured using Geiger–Müller counter to detect ionising radiation

5.     Radioactivity and concentration of antigens compared together shown in figure 5

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Advantages

Highly sensitive (Pelczar, M and Chan, E. 2010)

Disadvantages

Use of hazardous radiolabelled antigen

Require licence to use

Special waste disposable

Expensive (Bunko, H and Hisada K. 1976)

Requires trained personnel

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Advantages: (Novus Biologicals, n.a)

Easier procedure compared to IF

Easier lab reproduction

More resistant to light

Disadvantages: (Novus Biologicals, n.a)

Difficult to observe

Difficult to detect types of colour

Too much to visualise on one slide

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2. Procedure (Taylor and Phil 2013).

1.     Tissue fixation – to maintain tissue structure formalin – fixed and paraffin – embedded method

2.     Sectioning cut paraffin – embedded tissue blocks into thin sections, place on glass slides

3.     Antigen retrieval - ease of antigen expression, recover antigens changed by fixation

4.     Blocking – block endogenous enzymes such as peroxidase in kidney and liver get in the way of antibody binding visualization (Innova Biosciences 2014).

5.     Antibody labelling – Primary antibody binds to antigen; secondary antibody carrying enzyme binds to primary antibody

6.     Chromogen applied for visualisation under microscope

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3. Applications in Biomed (Duraiyan et al 2012)

Genetics – gene function

Neurogenetive disease – nerve cell roles development and death

Cancer markers – tumour identification via enzymes. Disease diagnosis, drug development at research. Malignant or benign tumour, stage and site of tumour

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2. Direct (Mohan et al 2008)
1. Biopsy sample from surgery
2. Sample snap frozen
3. Frozen sample cut on cryostat - placed on glass slide
4. Sample rinsed with PBS solution; dyed with FITC
5. Slides buffered in glycerine; observed under fluorescence microscope
Advantages:

-       Shorter staining times

-       Single step to labelling

-       Less cross reactivity

-       Less false positive results (Mohan et al 2008)

Disadvantages:

-       Less flexible with labelling

-       Expensive

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3. Indirect (Mohan et al 2008)
1. Substrate diluted at 1:10 ratio of patient serum for 30 minutes.

2. Rinse in PBS then add FITC with specific antibody conjugate

3. Rinse in PBS for 10 minutes; buffer with glycerine and observed under fluorescence microscope
Advantages:

More signalling because more than one antibody can attach to each primary (Kalyuzhny, A. 2016).

Less expensive

Available in many colours

High resolution

Disadvantages:

Cross reactivity possible (Kalyuzhny, A. 2016)

More steps involved

Flourescent dye can fade

(Kalyuzhny, A. 2016)

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3.     Applications in Biomed (Kumar et al 2009)

Observation of bacterial and parasitic samples

Antigens in frozen and fixed tissue

DNA sequence and chromosomal detection – presence or absence

Hormone detection

Skin, lung, muscle tissue examination

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Bunko, H and Hisada K.. (1976). [Advantages, disadvantages and selection methods of thyroxine measurement--comparison of CPBA and RIA]. Radioisotopes. 25 (10), p608-613.

Duraiyan, J., et al (2012). Applications of immunohistochemistry. J Pharm Bioallied Sci. p307-309.

Greb, C. (2012). Fluorescent Dyes [online]. Available at: http://www.leica-microsystems.com/science-lab/fluorescent-dyes/. [Accessed 29th October 2016].

Jones, R and Lopez, K. (2014). Human Reproductive Biology. 4th ed. China: Elsevier. p4-5.

Innova Biosciences. (2014). Getting started with Immunohistochemistry [online]. Available at: http://bitesizebio.com/20929/getting-started-with-immunohistochemistry/. [Accessed 29th October 2016].

Kalyuzhny, A. (2016). Immunohistochemistry: Essential Elements and Beyond. United States of America : Springer . p26-27 .

Kumar, G., et al (2009). Immunohistochemical (IHC) Staining Methods. 5th ed. North America: Dako. p61-65.

Mohan, K.H., et al (2008). Techniques of immunofluorescence and their significance. Indian J Dermatol Venereol Leprol. 74 (4), 415-419.

Novus Biologicals. (n.a). Immunohistochemistry (IHC) Detection [online]. Available at: https://www.novusbio.com/ihc-detection. [Accessed] 29th October 2016.

Pelczar, M and Chan, E. (2010). Microbiology:Application Based Approach. New Delhi: Tata McGraw Hill. p532.

Taylor, C and Phil, D. (2013). Immunohistochemical (IHC) Staining Methods. 6th ed. Denmark : Dako. p11-13.

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Determine levels of insulin in diabetics

Measure hormone concentration by the use of antibodies

Used in tests for endocrine disorders

Measured by use of antibody against hormone (Jones, R and Lopez, K. 2014).

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Method for analysing tissue samples to focus on antibodies specifically binding to antigens

For diagnosing cancer – staining required (Duraiyan et al 2012)

Observe tissue structure and parts of cell

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1. What is it?

Laboratory staining technique for research and diagnosis

Observation of antibodies bound to antigens in tissues using fluorescent dye – luminescence.

Molecules absorb light the excitation wavelength and emit this light at different lower energy wave length - emission wave length (Kumar et al 2009).

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Figure 4: Radioimmunoassay calibration curve shows radioactivity and antigen concentration comparison represented graphically – as unlabeled antigen increases, radio of bound radiolabeled antigen to free radiolabeled antigen decreases (Pelczar, M and Chan, E. 2010).

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Figure 5: [A] Radiolabeled antigens bind to antibodies

[B] Unlabeled and labelled antigen compete for free antibody binding sites (Pelczar, M and Chan, E. 2010).

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Figure 1: Drosophila embryo development visualisation by the used of FITC dye – highlighted in green (Greb 2012).

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Figure 2: Excitation and emission of light energy in fluorescence (Kumar et al 2009).

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Figure 3: Diagrammatic process of direct and indirect immunofluorescence differences.  (Kumar et al 2009)