Elisa

SHORT ELISA

1. Presented By Dr . V. RAMSEH , Assistant Professor & Head i/c Department of Botany Vivekananda College Tiruvedakam West – 625 234 [email protected] [phone] ENZYME LINKED IMMUNO SORBENT ASSAY (ELISA)

2. HISTORICAL BACKGROUND 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 ) & alkaline phosphatase Substrate: O-Phenyl- diamine - dihydrochloride for peroxidase P Nitrophenyl Phosphate- for Alkaline Phosphatase The enzyme acts as a catalyst to oxidize substrate in the presence of Hydrogen peroxide to produce a blue color.

4. PRINCIPLE 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.

5. TYPES OF ELISA

6. ADVANTAGES 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 . DISADVANTAGES Cross-reactivity may occur with the secondary antibody , resulting in nonspecific signal. An extra incubation step is required in the procedure .

7. ADVANTAGES Quick methodology since only one antibody is used. Cross-reactivity of secondary antibody is eliminated DISADVANTAGES 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.

8. 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.

9. APPLICATIONS Screening donated blood 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)

10. THANK YOU