Paper : 10: Module : 25: Clinical Immunoassays- ELISA, RIA, FACS,

Development Team

Principal Investigator: Prof. Neeta Sehgal Department of Zoology, University of Delhi

Co-Principal Investigator: Prof. D.K. Singh Department of Zoology, University of Delhi

Paper Coordinator: Prof. Anju Shrivastava Department of Zoology, University of Delhi

Content Writer: Dr. Divya Bajaj Hindu College, University of Delhi

Content Reviewer: Prof. Sukhmahendra Singh Banaras Hindu University

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

Description of Module

Subject Name ZOOLOGY

Paper Name Zool 010: Immunology

Module Name/Title Immunity in health and diseases

Module ID M25 Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence Immunolabels, ELISA, , immunoflorescence, FACS, Keywords , , indirect and direct detection, competitive, noncompetitive , isotope, reporter, , fluorochrome.

Contents

1. Learning Outcomes 2. Introduction 2.1. Immunoassays 2.2. Categories of Immunoassays 3. Visualizing the molecule of interest 3.1. Labels for Immunoassays 3.2. Indirect vs. Direct Detection methods 4. Clinical Immunoassays 4.1. -Linked Immunosorbent Assay  Variants of ELISA  Chemiluminescence  ELISpot Assay 4.2. Radioimmunoassay 4.3. Immunofluorescence 4.4. Flow Cytometry  FACS- Fluorescence activated cell sorter 5. Summary

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

1. Learning Outcomes

After reading this unit, you will be able to:

1. Define the basic characteristics of an . 2. Understand how a target molecule can be detected and quantitated. 3. How different types of labels can be employed to detect immune reactions. 4. How a wide array of information can be obtained relevant to the nature and localization of the target . 5. Differentiate between direct and indirect detection methods. 6. Basic principles and clinical applications of immunoassays.

2. Introduction

2.1. Immunoassays

An immunoassay isa biochemical test used to identify the presenceor amount of a particular moleculereferred to as an "analyte”, in a solution by combining it with an or an . The principal of immunoassays is formation of an immune complex involving the recognition and binding of an antibody to a specific molecule among a mixture of molecules. A key feature of all immunoassays is generation of a measurable signal in response to the binding. Immunoassays utilize a wide range of labels, some emit radiation, result in a visible colour change, fluoresce under light, or could be induced to emit light.

Detectable Label

Antibody

Analyte

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

Figure 1: Basic components of an Immunoassay. The analyte specifically binds to the antibody labeled with detectable label.

Examples of the application of immunoassay include:  Drug testing  Hormone testing (insulin in diabetic patients)  Bacterial or viral testing (AIDS, hepatitis)  Environmental testing (herbicides, pesticides)

Advantages of immunoassays are:  Inexpensive  Highly selective  Low limits of detection  High-throughput usually  Applicable to the determination of a wide-range of compounds

Ab* Ag Ag-Ab*

+

Labeled Antibody Analyte in Antibody-Analyte Reagent Specimen Complex

Ab Ag* Ag Ag*-Ab + Ag-Ab

+ +

Antibody Labeled Analyte in Antibody-Analyte Reagent Analyte Specimen Complex Reagent

Figure 2: A Detectable label can be attached to the antibody or antigen.Antigen antibody binding results in a complex formation and is detected with the help of a label.

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

2.2. Categories of Immunoassays

 Competitive –A competitive assay or limited reagent assay involves measurement of an unlabeled analyte or antigen by its ability to compete with the labelled antigen in the immunoassay. The assay mixture consists of saturated by labelled antibodies, hence higher the reduction in label at the end of assay, greater is the amount of antigen in the test sample.

 Non-Competitive One site Non-competitive - The unknown analyte in the sample are allowed to react with labelled antibodies. After the binding reaction is complete, unbound antibodies are washed away, and the bound labelled antibodies are measured as signals for the complexes formed. Therefore, intensity of the signal is directly proportional to the concentration of unknown antigen. Two site Non-competitive - An antibody adsorbed on the solid phase surface is allowed to interact with the test sample. The labeled antibodies are in excess in this system and bind specifically to their respective analyte. Subsequently, a second labeled antibody is added causing sandwiching of the target analyte. The quantitation of the labelled antibody helps in determining the concentration of the antigen since the two are directly proportional. The technique is also known as sandwich assay because the analyte is "sandwiched" between two antibodies.

 Heterogenous– Immunoassays involving the separation of bound Ab-Ag complex are known as heterogeneous immunoassays. The portion of antigen that combines with the antibody is isolated from the remaining unbound region after the reaction takes place. This separation is done via solid phase , liquid phase adsorption or precipitation.

 Homogenous –Homogenous assays are those which involve a change in the label as a result of the binding process, thereby allowing the binding to be evident without a separation step. 5

ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

Homogeneous methods do not involve the separation of the Ab-Ag complexes from the free Ag, and thus they are usually much easier and faster to perform. These assays are generally used for the measurement of small analytes such as therapeutic drugs.

Signal

3. Visualizing the Molecular of Interest

3.1. Labels for Immunoassays

Label is one of the reactant molecules participating in an immunoassay and in the process, it generates a measurable signal in solution. The interaction between a label and antibody or antigen molecule is generally chemical in nature. The different categories of labels used in immunoassays are:

1. - Enzymes aid in detection by generating a visible color change on reacting with substances which act as their substrates. Immunoassays that comprise of enzymes are referred to as enzyme-linked immunosorbent assays (ELISAs), or enzyme immunoassays (EIAs). In some assays, the enzymes react with reagents resulting in production of light or chemiluminescence. Enzymes commonly used in such reactions are (HRP), (AP) and glucose oxidase.

2. Radioactive Isotopes- Radioactive isotopes can also be used as labels for immunoassays and the radioactivity emitted by bound antibody-antigen complexes can be detected by various methods. Radioimmunoassays (RIA) are very sensitive, however they are considered potentially hazardous.

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

3. DNA Reporters- Real-time immunoquantitative PCR (iqPCR) assays use a DNA probe as label. This method combines real-time quantitative polymerase chain reaction (RT qPCR) and traditional immunoassay techniques.

4. Fluorogenic reporters- Protein microarrays often employ flourogenic reporters like phycoerythrin and rhodamine that help to visualize the completion of an immunoassay by generating change in colour.

Table 1: Detection of target molecules using different types of labels.Detection of antigen or antibody molecules can be done using different types of labels.

3.2. Indirect Vs. Direct Detection Methods

Antigens can be detected by ‘direct’ or ‘indirect’ association with a label in an immunoassay. Direct detection involves association of the label directly to the primary antibody by covalent bond formation; whereas in indirect detection methods the label is covalently attached to the secondary antibody, which then binds to the primary antibody during the assay. Indirect detection comprises of two parts: first, incubation with the unlabeled primary antibody, washing excess unbound antibody and second, addition of a labeled secondary antibody. After the excess secondary reagent is washed away, the amount of label associated with the primary antibody (via the secondary antibody) is quantified. Direct detection, however involves the prior covalent attachment of the label to the primary antibody allowing faster and simpler detection. The assay simplification achieved by direct detection tends to decrease variability, hence, improving data quality.

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

Despite the potential advantages of direct detection, many immunoassays still employ the principle of indirect detection. Chemical-modification of the primary antibody in direct detection could alter its antigen binding capacity. Indirect assays are also able to achieve higher sensitivity since each primary antibody contains several that combine with the labeled secondary antibody, resulting in signal amplification.

Table 2: Pros and Cons of Direct Vs Indirect Labeling. Both methods are used depending upon the desired reaction.

Method Pros Cons

 Immunoreactivity of the primary antibody  Faster process; uses one antibody. may be compromised as a result of Direct  Non-specific binding of secondary labeling. antibody eliminated.  Signal less intense.

 Enhanced sensitivity achieved as each  Nonspecific binding may occur during the primary antibody contains several epitopes addition of secondary antibody. Indirect that combine with the labeled secondary  Extra incubation and wash steps make the antibody, resulting in signal amplification. process time consuming.

4. Clinical Immunoassays

4.1. Enzyme-Linked Immunosorbent Assay

Enzyme-linked immunosorbent assay, commonly known as ELISA or EIA was first developed by Avramais (1966, 1969) and Pierce (1967).In this assay, an antibody coupled enzyme reacts with a colorless called a chromogenic substrate to generate a visible coloured reaction product. The enzymes commonly employed for ELISA, include alkaline phosphatase, horseradish peroxidase, and galactosidase. These assays possess high sensitivity and are safe and cost effective. The result generated from an ELISA assay could be qualitative identifying the presence or absence of a particular antigen molecule; semi-quantitative, analyzing relative antigen amounts in assay samples or quantitative, defining precise antigen concentrations with respect to a standard curve.

There are Numerous Variants of ELISA

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

ELISA assays can be performed in a variety of ways which allow both qualitative and quantitative analysis of either antigen or the antibody. ELISA can be used to identify the presence of antibody or antigen qualitatively. The unknown concentration of a sample can alternatively be determined by a curve based on known concentrations of antibody or antigen. The assay variants are described below -

Indirect ELISA

Indirect ELISA is used for both qualitative and quantitative measurements of antibodies. The procedure includes addition of the sample solution containing primary antibody (Ab1) to a microtiter well pre-coated with the antigen, such that the antibody would react with this well bound antigen. Unbound antibody is washed away and this is followed by detection of the antibody bound to the antigen with the help of an enzyme-conjugated secondary anti-isotype antibody (Ab2) which specifically binds to the primary antibody Ab1.Unbound secondary antibody is also washed away followed by addition of substrate for the enzyme. The coloured reaction product is analyzed spectrophotometrically by plate readers.

Figure 3: Indirect ELISA - Antigen is immobilized on the surface and sample is added, if antibodies specific to the antigen of interest is present binding would occur and visualized with the help of enzyme conjugated secondary antibody. Immunology, Sixth edition, Kuby, 2007, WH Freeman and company.

Indirect ELISA is preferred for detecting the presence of serum antibodies against human immunodeficiency virus (HIV), the causative agent of AIDS. The recombinantenvelope and core proteins of HIV are used as antigens plated on to microtiter wells.

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

Sandwich ELISA

Sandwich ELISA is used for quantitative or qualitative analysis of antigens. The basis of this technique remains the antigen-antibody interaction however;the antibody instead of the antigen is immobilized on the microtiter well. An antigen sample is then added to the well pre-coated with theimmobilized antibody. The excess or unbound antigen is washed off using buffers, followed by addition of a second enzyme-linked antibody specific to a second on thebound antigen. Unbound secondary antibody is then washed off and a substrate corresponding to the enzyme on the secondary antibody this added, and the coloured reaction product analyzed spectrophotometrically.

Figure 4: Sandwich ELISA – the antigen of interest is sandwiched between primary antibody immobilized on solid surface and enzyme conjugated secondary antibody. Immunology, Sixth edition, Kuby, 2007, WH Freeman and company.

Competitive ELISA

Competitive ELISA is also a variant technique for the quantitation of antigen. The procedure consists of a pretreatment step where the antibody isincubated in solution with a sample containing the antigen. A microtiter plate coated with the same antigen is then incubated with the previously procured antigen-antibody mixture. Greater the amount of antigen in the sample, lesser would be the amount of free antibody available to bind to theantigen-coated well. On addition of an enzyme- conjugated secondaryantibody (Ab2) specific for the isotype of the primaryantibody, the amount of primaryantibody immobilized on the well can be determined like in an indirect ELISA. This competitive interaction suggests that higher the amount ofantigen in the original sample, the lower would be the value of absorbance.

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

Figure 5: Competitive ELISA - Antigen-antibody mixture is added in addition to the free antibodies and incubated with antigen coated wells. Enzyme conjugated secondary antibodies when allowed to react with substrate generate coloured product which is quantitated by measuring absorbance.Immunology, Sixth edition, Kuby, 2007, WH Freeman and company.

Substrate

Substrate

Substrate Substrate Secondary antibody conjugate

Primary Capture antibody antibody conjugate

Direct ELISA Indirect ELISA Sandwich ELISA Competitive ELISA

Figure 6: Comparison between different types of ELISA. This overview helps to visualize the differences between different ELISA variants. A particular type of assay can be selected depending upon specific interests.

Chemiluminescence

Chemiluminescence is an ELISA variant where an enzyme substrate reaction produces a byproduct that emits light (luxogenic) instead of developing a visible color. Luminescence is defined as emission of light from a body as it returns from an electronically excited state to ground state. The different types of luminescence like bioluminescence, chemiluminescence, photoluminescence differ in the way this excited state is achieved. Chemiluminescence consists of measurement of light duringa chemical reaction. It is a convenient andhighly sensitive alternative to absorbance measurements

inELISA assays. For example, oxidation of the compound luminol byH2O2 and the enzyme horseradish peroxidase (HRP) produceslight: 11

ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

luminol _ H2O2 Ab-HRP _ Ag → Ab-HRP-Ag →light

The advantage of chemiluminescence assays over chromogenic reactions is enhanced sensitivity. The detection limit of a luxogenic substrate is at least ten-fold higher than a chromogenic one, and with the help of enhancing agents, it could be increased to more than 200-fold.

Figure 7: Chemiluminescent Sandwich ELISA assay

Principle: A pre-coated with capture antibody is added with sample, specific analyte if present is bound by the antibody and unbound substances are washed away. A secondary HRP-labeled antibody (detection antibody) is added which binds to the captured analyte. Unbound detection antibody is washed away. Enhanced Luminol substrate is added and light is produced in proportion to the amount of analyte present in the sample and the intensity of the light emitted is measured.https://www.rndsystems.com/products/quantiglo- chemiluminescent-sandwich-elisas

ELISpot Assay

Cecil Czerkinsky’s group in Gothenburg, Sweden in 1983 first developed the enzyme linked immunospot (ELISpot) assay, in order to detect antigen-specific antibody producing cells.This modified ELISA assay, helps in identification and quantitation of the number of cellsin a population that can produce antibodies in response to aparticular antigen or vice versa. The procedure involves adsorption of the plates with antigen called the capture antigenfollowed by reaction with the antibodyof interest or with the antibody (capture antibody) specific to the target antigen. Apopulation of cells to be analyzed is thenadded in the form of a suspension on to the treated plates and incubated. The cells collect at the surface of the plates and bind to the secreted substances specific to these 12

ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

capture molecules,producing a ring ofantigen-antibody complexes around the cells secreting the molecule of interest. Unbound molecules are washed off and anenzyme-linked antibody specific for the secreted antigen is allowed to bind. The assay is subsequently developed by chromogenic or chemiluminescent substrates identifyingeach antibody or antigen producing cell indicated by colour or light. A substrate is selected such that a precipitate is produced instead of a soluble product, displaying the result in the form of visible spots corresponding to each antigen-secreting cell. The ELISpot technique is commonly employed for measurement of ; and is suitable for almost any secreted protein where single-cell analysis is of interest. The method was originally developed to quantify immunoglobulin-secreting cells.ELISpot is capable of detecting a single antigen positive cell within a population of cells, having a detection limit of one in one million cells.

Figure 8: ELISPOT Assay: modified ELISA assay, where the antigen antibody reaction is detected by formation of coloured spots. https://www.rndsystems.com/products/elispot-assay-principle

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

4.2. Radioimmunoassay - RIA

Radioimmunoassay(RIA) one of the most sensitive techniques for detecting antigen orantibody was first reported by S. A. Berson and Rosalyn Yalow in 1960, in order toanalyze the levels ofinsulin - anti-insulincomplexes in diabetics. This was the first attempt for detection of blood hormonesby an in- vitro assay.The technique demonstrates high sensitivityand is capable of quantitating hormones, serum proteins, drugs, and vitamins at concentrations as low as 0.001 micrograms per milliliter. The basic principle of this technique is competitive binding between the radiolabeledand unlabeled antigen to a high-affinityantibody. First the antibody is allowed to interact with the radiolabeled antigen saturating the antigen-binding sites of theantibody. This is followed by addition oflarge amounts of sample containing unknown amount of unlabeled antigen. The available binding sites on the antibody are available to both the labeled and unlabeled antigens as the antibody is unable to distinguish between the two. The labeled antigen is progressively displaced from the antibody binding sites with increasing amount of the unlabeled antigen. This reduction in the amount of radiolabeled antigen bound to the specific antibody on increasing antigen concentration in the unknown sample is measured in order to quantitate antigen concentrations in the test sample.

The primary step for this assay is to ascertain the amount of antibodyneeded to saturate 50% - 70% of a specific quantity of radioactiveantigen in the test mixture. The antibodyto antigen ratiois taken such that the labeled antigen displays more number of epitopes than the totalnumber of antibody binding sites. This ensures competitive binding between unlabeled antigen added to the mixture and the radiolabeledantigen against the limited supply of antibody. The bound labeled antigen is quantitated by precipitating the Ag-Ab complex and segregating it from free antigen; and eventually the radioactivityof the precipitate is measured.

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

Radioactive antigen Y

First antibody

Add unlabeled antigen ( )

Radioactive antogen ( ) displaced by unlabeled antigen ( ) Y

Precipitate Ag-Ab complexes with Anti-immunoglobulin second antibody

Radioactivity of supernatent - Free antigen

Radioactivity of precipitate - Bound antigen

Second antibody Y

Figure 9: Radioimmunoassay (RIA): Based on competitive binding of radiolabeled and unlabeled antigen to a high-affinity antibody.

4.3. Immunofluorescence

Albert Coons first demonstrated the labeling of antibodies with fluorescent molecules in 1944. These molecules possess the inherent property of absorbing light of a particular wavelength (excitation) and emitting light of another wavelength. Antibodies tagged with a fluorescent dye, or fluorochrome, can be identified by emission of colored light when excited by light of a specific wavelength, when these are a part of immune complexes. This technique also allows detection of antibodies bound to antigen epitopes in cell cultures or tissue sections. Molecules with luminescent properties emit light of a different wavelength on absorbing light of a particular wavelength. Fluorescent materials give off light very promptly due to their atomic structure. The light emitted from luminescent objects can be visualized using a fluorescence microscope equipped with a UV light source. These fluorescent probes are conjugated to the Fc arm of an antibody molecule ensuring that its specificity is not affected. Commonly used fluorochromes are:

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

 Fluorescein is the most frequently used organic label dye for immunofluorescence. It absorbs blue light (490 nm) and emits a higher wavelength intense yellow-green fluorescence (517 nm).

 Rhodamine is another organic dye, absorbing light in the yellow-green range (515 nm) and emitting a deep red fluorescence (546 nm). Two-color immunofluorescence assays can be performed using a combination of these two dyes simultaneously as rhodamine emits fluorescence at a longer wavelength than fluorescein. Spatial distribution and comparative assays for two antigens are performed in a single experiment where an antibody specific to one determinant is tagged with fluorescein, and a second antibody recognizing another antigen is labeled with rhodamine. The co-localization of the fluorescein-tagged antibody visualized by its yellowgreencolor, is discretely distinguishable from the red color emitted where the rhodamine- tagged antibody is bound.

 Phycoerythrin an efficient absorber of light (~30-fold greater than fluorescein) and a brilliant emitter of red fluorescence, is also widely used as an immunofluorescence label.

Applications of immunofluorescence can be wide range, starting with identification of a number of subpopulations of cells in culture, identifying bacterial species, detecting Ag-Ab complexes in disease conditions, detection of complement components, as well as localizing and staining of hormones and other subcellular molecules in situ. It also finds use in analysis of cells in suspension, cultured cells, tissue, beads and microarrays for the detection of specific proteins. A very important application of immunofluorescence is tissue or cell specific antigen localization. The target antigens can be localized in cells or tissues andvisualized by fluorescence microscopy thus, making it a potent tool for associating the molecular architecture of tissues and organs to gross anatomy and physiology.

4.4. Flow Cytometry

Flow cytometry aims at automation of the process of cell-type segregation of immune stained cells and their subsequent analysis. The fluorescent antibody techniques are extremely efficient qualitative tools, but they do not permit quantitation of signals. The flow cytometer utilizes a laser beam and light detector for measuring cell signals in suspension hence eliminating this drawback. Each time the laser beam passes through a cell, the light coming from the deflector is deflected from its path, and this intermittent disconnect of the laser signal is recorded. The cells in suspension are tagged with a fluorescent label by direct or indirect immunofluorescence methods. This is followed by analysis of these cells on the flow cytometer. Cells bound to fluorescently labeled antibodies get excited on being hit with the laser beam and emit light which is subsequently recorded by a detector unit installed at a

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

right angle to the laser beam. These signals are directed towards a computer which generates plots of cell number vs. their fluorescence intensities. Advanced instruments are also involved in cell sorting into different locations according to their fluorescence intensity profiles. The output investigates the number and type of every cell in a population that binds fluorescently tagged antibodies.

Appropriately treated blood samples when combined with a fluorescently tagged antibody are employed for flow cytometric analysis, for yieldingvaluable information like:

 A specific cell type can be identified using a fluorescent antibody targeted against a cell surface antigen, and the percentage of this cell type in the total population of cells calculated.  The distribution of cells in a population of interest can be identified with respect to antigen densities reflected by fluorescence intensitiesin cell populations. The significance of this can be understood as the same cell type could be expressing variable quantities of antigen during different developmental or physiological states.  An estimate of cell size can also be done from analysis of the light-scattering ability of different cells in the cell population under observation.

FACS: Fluorescence-Activated Cell Sorter

Fluorescence-Activated Cell Sorting is a technique based on the principle of flow cytometry where cell sorting is performed on the basis of their protein constitution. It finds excessive application in immunology, where blood cell counts are measured in patients with HIV and leukemia, for diagnosis of the disease diagnosis as well as monitoring the patient’s response to treatment. Cell sorting is also effective in understanding the functions of drugs, development of drug resistance, detection and identification of viral antigensand development of new drugs for various disease conditions. The FACS method can detect cells with as few as 3,000 molecules of fluorescent dye bound to each cell. Further, the cell size is also measured by means of correlated light-scattering; such that the cells falling within particular ranges of both size and fluorescence can be separated.

All cells in a multicellular organismcontain identical DNA but tremendous variations are present at the protein level.Therefore this principle is utilized to separate cells that are phenotypically distinct. Moreover, FACS can quantitate the number of cells expressing the proteins of interest, and also the amount of protein expressed.

The fluorescence-activated cell sorter can promptly segregate the cells in suspension on the basis of size and the color of their fluorescence. The procedure followed is:

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

 Cells are labeled with a fluorescent dye and the cell suspension is projected in the form of a thin stream. The coupled dye combines with the cells coated with the respective antigen.  The stream of cells is projected through a nozzle vibrating at ~ 40,000 cycles per second hence disrupting it into 40,000 distinct droplets per second, out of which some may contain a cell.  A laser beam is directed on the stream just before it bursts into droplets. Fluorescence is therefore detected using a photocell as each labeled cell passes across the beam.  The output from the two detectors are analyzed for the desired criteria set for size and fluorescence, and a corresponding electrical charge ( + or - ) getsassigned to the stream.  The charged droplets are made to pass between a pair of charged metal plates and thus get attracted to oppositely charged plates therefore getting sorted accordingly, while uncharged droplets are eliminated.

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

Figure 10: Flourescence activated cell sorter : FACS – Basic procedure followed for cell sorting is depicted here. Cell types are identified and sorted according to their protein contents.

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence

One of the commonly used clinical applications of flow cytometry is to identify the type and number of each of the WBCs in blood samples. It has been successfully used in the detection and classification of leukemias by identification of the cell types involved, making discrete detection of the neoplastic cells an important part of clinical application. Similarly, the rapid measurement of T- cell subpopulations, a reliable prognostic indicator in AIDS, is done by flow cytometric analysis.

5. Summary

1. The key features of an immunoassay include formation of an immune-complex and production of a measurable signal in response to the binding which can be detected and analysed. 2. Immunoassays employ a wide range of labels, some emit radiation, produce a visible colour change, fluoresce under light, or can be induced to emit light. 3. A competitive assay or limited reagent assay is one where the amount of unlabeled analyte or antigen is measured by its ability to compete with the labelled antigen in the immunoassay. 4. The target cell type can be identified using a fluorescent antibody specific for a cell surface antigen, and the percentage of this cell type in the total cell population of interest can be identified. 5. Luminescence is defined as emission of light from a body as it returns from an electronically excited state to ground state. 6. All cells in a multicellular organism contain identical DNA but tremendous variations are present at the protein level. Therefore, this principle is utilized to separate cells that are phenotypically distinct by the FACS technique.

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence