Immunosorbent assay: ELISA

ELISA (enzyme-linked immunosorbent assay) is an immunological assay designed for detecting and quantifying soluble substances – such as peptides, proteins, antibodies and hormones – in biological samples. In an ELISA, the antigen (target macromolecule) is immobilized on a microplate (usually a 96-well plate). This is then complexed with a specific antibody that is linked to an enzyme. Commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Detection is achieved by measuring the activity of the enzyme via incubation with the appropriate substrate to produce a measurable product. The choice of substrate depends upon the required assay sensitivity and the instrumentation available for signal detection (spectrophotometer, fluorometer or luminometer). The ability to use high-affinity antibodies and wash away non-specific bound materials makes ELISA a simple yet powerful tool for measuring specific analytes within a crude preparation.