ELISA

Principle

An enzyme-linked antibody detects antigen or antibody bound to a solid phase. The enzyme converts a colourless substrate to a coloured product; colour intensity is proportional to the amount of target.

Common formats

• Indirect: antigen on the plate detects patient antibody (e.g. HIV antibody screening).
• Sandwich: capture antibody on the plate detects antigen (e.g. HBsAg).
• Competitive: patient antigen competes with labelled antigen; more colour = less antigen.

General procedure

• Add sample to coated wells and incubate; wash.
• Add enzyme conjugate (e.g. HRP); incubate; wash thoroughly.
• Add substrate (e.g. TMB); stop with acid and read absorbance (450 nm).

Quality control

• Run negative, positive and cut-off calibrators on every plate.
• Poor washing is the commonest cause of false positives.