ELISA and its advantages

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying soluble substances such as peptides, proteins, antibodies, and hormones. Other names, such as enzyme immunoassay (EIA), are also used to describe the same technology.
The enzyme-linked immunosorbent assay is a commonly used analytical biochemistry assay, first described by Engvall and Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay to detect the presence of a ligand in a liquid sample using antibodies directed against the protein to be measured.

1. ELISA Presented by: Farhana Akter Joty ID: 193-46-258

2. Prior to the development of the EIA/ELISA, the only option for conducting an immunoassay was radio immunoassay, 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 . History

3. Antibody Antigen Enzyme: Horse Radish Peroxidase ( HRP), alkaline phosphate ( AP). 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 . Components of ELISA

4. The basic enzyme-linked immunosorbent assay (ELISA), or enzyme immunoassay (EIA), is distinguished from other antibody-based assays because separation of specific and non-specific interactions occurs via serial binding to a solid surface, usually a polystyrene multiwell plate, and because quantitative results can be achieved. The ELISA procedure results in a colored end product which correlates to the amount of analyte present in the original sample. Introduction to ELISA

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. The ELISA assay yields 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 result

7. Semi- quantitative : ELISAs can be used to compare the relative levels of antigen in assay samples, since the intensity of signal will vary directly with antigen concentration. The standard curve : ELISA data is typically graphed with optical density vs log concentration to produce a sigmoidal curve as shown in Figure. 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. This can be done directly on the graph or with curve fitting software which is typically found on ELISA plate readers. Cont..

8. Cont.. Figure : A typical ELISA curve

9. 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) Application