Elisa AND ITS APPLICATION

ELISA AND ITS APPLICATION

1. ELISA (ENZYME LINKED IMMUNOSORBENT ASSAY) PRESENTED BY RAJPAL CHOUDHARY

2. HISTORY Prior to the development 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 A 96 - 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 principle of 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 in sample substrate Enzyme Colored product Primary antibody Secondary antibody

7. TYPES OF ELISA INDIRECT ELISA DIRECT ELISA SANDWICH ELISA COMPETETIVE ELISA NON -COMPETETIVE ELISA

8. INDIRECT ELISA Antigen is 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 INDIRECT DETECTION Wide variety of labeled secondary antibodies are available commercially. Versatile, since many primary antibodies can be made in one species and the s ame 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 Apply a 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 DIRECT DETECTION Quick methodology since only one antibody is used. Cross-reactivity of secondary antibody is eliminated.

13. DISADVANTAGES OF DIRECT DETECTION 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 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 phase coated 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 for complex ( 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 assay yields 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 data is 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 donated blood for evidence of viral contamination by HIV-1 and HIV-2 (presence of anti-HIV antibodies) H epatitis C (presence of antibodies) H epatitis 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)

20. THANK YOU