Know the Science behind detecting antibodies
By Dr. Modala Mallesh Hyderabad: This article is in continuation of the previous articles that help you understand what constitutes the biological sciences and its varied branches/ concepts. Today, we would be discussing the fifth part of the Biomedical technology with a focus on Enzyme-linked immunosorbent assay (ELISA). ELISA (Enzyme-linked immunosorbent assay) ELISA is […]
By Dr. Modala Mallesh
Hyderabad: This article is in continuation of the previous articles that help you understand what constitutes the biological sciences and its varied branches/ concepts. Today, we would be discussing the fifth part of the Biomedical technology with a focus on Enzyme-linked immunosorbent assay (ELISA).
ELISA (Enzyme-linked immunosorbent assay)
ELISA is the acronym (short form) of Enzyme-linked immunosorbent assay.
It is a biochemical procedure to detect antigens/antibodies in a given sample.
All microbes have at least an antigen that is unique.
This antigen can be purified and used to generate specific monoclonal antibodies (MAB). Thus, MABs are made available for this procedure.
Both the antibodies and the purified antigens provide effective diagnostic tools.
It is a method used mainly to detect the presence of specific antibodies/antigens in a sample of serum, urine etc.
It is the first and most basic tool to determine if an individual is positive/ negative for a particular pathogen such as HIV.
It is a useful tool both for determining serum antibody concentrations (such as antibodies produced in a person infected by pathogens such as HIV) and also for detecting the presence of specific antigens and hormones such as human chorionic gonadotropins (HCGs).
Requirements for ELISA:
A microtitre plate
A purified antibody (for detecting a specific antigen).
A purified antigen (for detecting a specific antibody).
An enzyme that catalyses the production of colour from a chromogenic substance (mostly peroxidase/ alkaline phosphatase/ beta galactosidase).
A buffer (wash) fluid to remove unbound substances in the well.
A substrate (chromogenic substance)
A spectrophotometer to measure the intensity of the colour of the substrate.
Note: Monoclonal antibodies are used as
1. Primary antibodies (which react with the antigens of interest).
2. Secondary antibodies (which react with the primary antibodies).
ELISA in clinical immunology:
ELISA is a fundamental tool of clinical immunology, and the purpose of an ELISA is to determine if a particular protein is present in a sample, such as serum of a person affected by pathogen and if to how much.
Types of ELISA:
ELISA is of two types:
a. Direct ELISA- ELISA used to detect antigens
b. Indirect ELISA – ELISA done to detect antibodies.
Direct ELISA:
Direct ELISA is performed by using primary antibodies (antibodies against the antigen of interest) attached/ adsorbed on the solid surface of the well on the titre plate.
This antibody has affinity for the antigen of interest (antigen for whose detection this test is being conducted).
It is of two types
A. Sandwich Assay
B. Competitive Assay
Indirect ELISA:-
It is used to detect antibodies
The blood of the person undergoing the assay (for example the HIV test) is allowed to clot and the clear serum with antibodies (called primary antibodies) is obtained.
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