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Elisa AND ITS APPLICATION
The ELISA (enzyme-linked immunosorbent assay) is a test that uses antibodies and color change to identify a substance. It involves using an enzyme to detect antigen-antibody binding, which converts a colorless substrate into a colored product. There are several types of ELISA including indirect, direct, sandwich, and competitive ELISA. ELISA can provide quantitative or qualitative results and has applications like screening donated blood and measuring hormone levels.
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Elisa AND ITS APPLICATION
- 1. ELISA (ENZYME LINKED IMMUNOSORBENT ASSAY) PRESENTEDBY RAJPAL CHOUDHARY
- 2. HISTORY Prior to thedevelopment of the EIA/ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using radioactively-labeled antigens or antibodies. Avrameas (1966, 1969) and Pierce (1967) developed methods to chemically link antibodies to biological enzymes whose activities produce a measurable signal with solutions containing appropriate substrates. This signal has to be associated with the presence of antibody or antigen .
- 3. COMPONENTS OF ELISA Antibody Enzyme:Horse Radish Peroxidase (HRP) MW 44, 000, glycoprotein with 4 lysine residues. Substrate: TMB (3,3',5,5', tetramethylbenzidine) The enzyme acts as a catalyst to oxidize substrate in the presence of Hydrogen peroxide to produce a blue color.
- 4. INTRODUCTION TO ELISA A96 - well microtiter plate being used for ELISA. A test that uses antibodies and color change to identify a substance. ELISA involves at least one antibody with specificity for a particular antigen.
- 5. The basic principleof an ELISA is to use an enzyme to detect the Ag-Ab binding (antigen- antibody binding). The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag:Ab binding. PRINCIPLE
- 6. Different antigen insample substrate Colored product Primary antibody Secondary antibody
- 7. TYPES OF ELISA INDIRECT ELISA DIRECT ELISA SANDWICH ELISA COMPETETIVE ELISA NON -COMPETETIVE ELISA
- 8. INDIRECT ELISA Antigenis added to plate. Added buffer. Suitable primary antibody is added. Secondary antibody- HRPO is then added which recognizes and binds to primary antibody. TMB substrate is added, is converted to detectable form.
- 9. ADVANTAGES OF INDIRECTDETECTION Wide variety of labeled secondary antibodies are available commercially. Versatile, since many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection. Immunoreactivity of the primary antibody is not affected by labeling. Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification.
- 10. DISADVANTAGES OF INDIRECT DETECTION Cross-reactivity may occur with the secondary antibody, resulting in nonspecific signal. An extra incubation step is required in the procedure.
- 11. DIRECT ELISA Applya sample of known antigen to a surface. Enzyme linked primary antibody is applied to the plate. Washed, After this wash, only the antibody-antigen complexes remain attached. Apply a substrate which is converted by the enzyme to elicit a chromogenic signal.
- 12. ADVANTAGES OF DIRECTDETECTION Quick methodology since only one antibody is used. Cross-reactivity of secondary antibody is eliminated.
- 13. DISADVANTAGES OF DIRECTDETECTION Immunoreactivity of the primary antibody may be reduced as a result of labeling. Labeling of every primary antibody is time-consuming and expensive. No flexibility in choice of primary antibody label from one experiment to another.
- 14. SANDWICH ELISA 1. a.Plate is coated with suitable antibody. b. Buffer is added. 2. Sample is added to plate so antigen is bounded by capture antibody. 3. A suitable biotin labeled detection antibody is added to plate. 4. Enzyme HRPO is added and binds the biotin labeled detection antibody. 5. TMB substrate is added and converted by HRPO to colored product.
- 15. COMPETETIVE ELISA Solid phasecoated with antibody Add unknown amount of unlabeled antigen and known amount of labeled antigen Free and labeled antigen are captured Color formation by oxidation of substrate into a colored compound
- 16. ADVANTAGES Suitable forcomplex (crude or impure) samples, since the antigen does not require purification prior to measurement. DISADVANTAGES Each antigen may require a different method to couple it to the enzyme.
- 17. The ELISA assayyields three different types of data output: Quantitative: ELISA data can be interpreted in comparison to a standard curve in order to precisely calculate the concentrations of antigen in various samples. Qualitative: ELISAs can also be used to achieve a yes or no answer indicating whether a particular antigen is present in a sample, as compared to a blank well containing no antigen or an unrelated control antigen. ELISA RESULTS
- 18. ELISA datais typically graphed with Optical density Vs Log concentration to produce a sigmoidal curve. Known concentrations of antigen are used to produce a standard curve and then this data is used to measure the concentration of unknown samples by comparison to the linear portion of the standard curve.
- 19. APPLICATIONS Screening donatedblood for evidence of viral contamination by HIV-1 and HIV-2 (presence of anti-HIV antibodies) Hepatitis C (presence of antibodies) Hepatitis B (testing for both antibodies and a viral antigen) Measuring hormone levels HCG (as a test for pregnancy) LH (determining the time of ovulation) TSH, T3 and T4 (for thyroid function)
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