The document summarizes the development and basic principles of enzyme-linked immunosorbent assays (ELISAs). It explains that ELISAs were developed as an alternative to radioimmunoassays, which used radioactive labels. ELISAs link antibodies to enzymes, which catalyze color changes in substrates to signal the presence of antigens. The ELISA procedure results in a colored end product corresponding to antigen levels. ELISAs can provide quantitative, qualitative, or semi-quantitative results by comparing sample readings to a standard curve of known antigen concentrations. Common applications of ELISAs include screening blood donations and measuring hormone levels.

1. 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 .

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.

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.

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.

7. 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.

8.  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.

9. Figure : A typical ELISA curve

10.  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)