SHORT ELISA

1. Presented By
Dr. V. RAMSEH,
Assistant Professor & Head i/c
Department of Botany
Vivekananda College
Tiruvedakam West – 625 234
ramesh.vnr09@gmail.com
+91 9842629245
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.

5. 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.

7. TYPES OF ELISA

9. 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.

11. 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.

13. 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.

14. 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)

15. THANK YOU