Question
ELISA
Answer
ELISA (Enzyme-Linked ImmunoSorbent Assay) is a serological technique that detects and quantifies a specific antigen or antibody using an enzyme-labelled reagent, with a colour change (measured spectrophotometrically as optical density) indicating and quantifying a positive reaction.
Principle: one reactant (antigen or antibody) is fixed to a solid-phase support (typically microtiter plate wells); 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, binding the captured target; after further washing, a chromogenic substrate is added, and the enzyme converts it to a coloured product, whose intensity is proportional to the amount of target present.
Types:
- Direct ELISA — enzyme-labelled antibody binds directly to antigen coated on the plate.
- Indirect ELISA — used to detect antibody; antigen is coated on the plate, patient serum is added, 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 sample is added, followed by a second, enzyme-labelled detection antibody binding a different epitope — high specificity.
- Competitive ELISA — labelled and unlabelled antigen compete for a limited amount of antibody; signal is inversely proportional to antigen in the test sample.
Applications: serodiagnosis of infections (HIV, hepatitis B/C, dengue), detection of microbial antigens/toxins, hormone assays (beta-hCG pregnancy testing), allergen-specific IgE testing, and CD4 count-related assays — valued for high sensitivity, ability to process many samples simultaneously, and suitability for automation and large-scale screening.