Enzyme-linked immunosorbent assay (ELISA) is a plate-based immunoassay used to detect and quantify specific proteins, antibodies, hormones, and other molecules. It combines the specificity of antibodies with the sensitivity of enzyme-based detection.
How ELISA Works
- Coating
A microtiter plate is coated with an antigen or capture antibody. The target molecule binds to the surface through passive adsorption. The plate is incubated overnight and then washed to remove unbound material.
- Blocking
A blocking solution containing an irrelevant protein such as BSA or casein is added to the wells. This covers any remaining binding sites on the plate surface, preventing non-specific binding in subsequent steps.
- Detection Antibody
A detection antibody conjugated to an enzyme—most commonly horseradish peroxidase (HRP) or alkaline phosphatase (AP)—is added. In a sandwich ELISA, a primary antibody binds the target, and an enzyme-linked secondary antibody binds the primary.
- Signal Generation
A substrate for the enzyme is added. The enzyme converts the substrate into a colored, fluorescent, or luminescent product. The reaction is allowed to proceed for a set time and then stopped.
- Measurement
The signal is measured using a plate reader. The intensity of the signal is proportional to the amount of target molecule in the sample. A standard curve using known concentrations allows quantification.
- ELISA Formats
- Direct ELISA: Antigen is coated, and an enzyme-linked antibody detects it directly.
- Indirect ELISA: Antigen is coated, a primary antibody binds, and an enzyme-linked secondary antibody detects it.
- Sandwich ELISA: A capture antibody is coated, the antigen binds, and a detection antibody completes the sandwich.
- Competitive ELISA: Signal decreases as target concentration increases.
