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

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ZOOLOGY Immunology Clinical Immunoassays- ELISA, RIA, FACS, Immunofluorescence Paper : 10: Immunology Module : 25: Clinical Immunoassays- ELISA, RIA, FACS, immunofluorescence 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 1 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, Radioimmunoassay, immunoflorescence, FACS, Keywords Flow cytometry, chemiluminescence, indirect and direct detection, competitive, noncompetitive assay, isotope, reporter, elispot, 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. Enzyme-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 2 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 Immunoassay. 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 antigens. 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 antibody or an antigen. 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 3 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. 4 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 antibodies 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 adsorption, 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- 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 horseradish peroxidase (HRP), alkaline phosphatase (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. 6 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. 7 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 epitopes 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
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