An enzyme-linked immunosorbent assay (ELISA) is a laboratory technique used to detect and measure the presence of specific substances, such as antibodies, antigens, proteins, hormones, or other molecules, in a sample. ELISA is widely used in medical diagnostics, research laboratories, and various fields of biology and biochemistry.
The basic principle of ELISA involves the use of specific antibodies that can recognize and bind to the target molecule of interest. The assay typically involves the following steps:
Coating: A microplate is coated with a capture antibody that specifically binds to the target molecule (e.g., antigen) present in the sample.
Blocking: The microplate is then treated with a blocking agent to prevent nonspecific binding of other molecules to the plate surface.
Sample Incubation: The sample containing the target molecule is added to the microplate and allowed to incubate. If the target molecule is present in the sample, it will bind to the capture antibody immobilized on the plate.
Detection: After washing away unbound substances, a detection antibody conjugated with an enzyme (such as horseradish peroxidase or alkaline phosphatase) is added. This detection antibody binds to a different epitope on the target molecule, forming a "sandwich" complex.
Substrate addition: The microplate is washed again to remove unbound detection antibodies, and then a substrate specific to the enzyme is added. The enzyme catalyzes a color-producing reaction with the substrate.
Signal measurement: The intensity of the color produced is measured spectrophotometrically. The intensity of the color is directly proportional to the amount of target molecule present in the sample.
ELISA assays can be performed in various formats, including direct, indirect, sandwich (capture), and competitive assays, depending on the specific application and the nature of the target molecule being detected. ELISA is known for its high sensitivity, specificity, and versatility, making it a valuable tool in biomedical research and diagnostics.