Question
ELISA
Answer
ELISA (Enzyme-Linked ImmunoSorbent Assay) is a widely used serological technique that detects and quantifies a specific antigen or antibody in a test sample using an enzyme-labelled reagent, with a colour change (measured spectrophotometrically) indicating and quantifying a positive reaction.
Principle: one of the reactants (antigen or antibody) is fixed to a solid-phase support (typically the wells of a microtiter plate); the sample is added and allowed to bind specifically if the target is present; unbound material is washed away; an enzyme-conjugated detection reagent is added, which binds the captured target; after further washing, a chromogenic (colour-producing) substrate is added, and the enzyme converts it to a coloured product, whose intensity (measured as optical density) is proportional to the amount of target present.
Common formats:
- Direct ELISA — enzyme-labelled antibody binds directly to the antigen coated on the plate.
- Indirect ELISA — used to detect antibody in patient serum; antigen is coated on the plate, patient serum is added (if specific antibody is present, it binds), followed by an enzyme-labelled anti-human-immunoglobulin secondary antibody.
- Sandwich ELISA — used to detect antigen; a capture antibody is coated on the plate, the test sample (containing antigen) is added, followed by a second, enzyme-labelled detection antibody that binds a different epitope on the same antigen, “sandwiching” it between the two antibodies — provides high specificity.
- Competitive ELISA — labelled and unlabelled antigen compete for a limited amount of antibody; signal is inversely proportional to the amount of antigen in the test sample.
Applications: serodiagnosis of infections (HIV, hepatitis B/C, dengue, etc.), detection of microbial antigens/toxins, hormone assays, and allergen-specific IgE testing — valued for its high sensitivity, ability to process many samples simultaneously, and suitability for automation.