Immunoassays are laboratory tests that leverage the specific interaction between antibodies and antigens to detect or quantify substances in a sample. The Enzyme-Linked Immunosorbent Assay (ELISA) is a highly sensitive and versatile technique. It is a fundamental method in scientific research and medical diagnostics, identifying or measuring specific molecules like proteins, hormones, or antibodies within various biological samples.
What is ELISA?
ELISA is a laboratory technique used to detect and measure specific substances in liquid samples. Its core principle involves using antibodies, proteins that recognize and bind to specific targets (antigens), and enzymes, which are biological catalysts. This combination allows for the detection of either antigens or antibodies within a sample.
The method is “enzyme-linked” because an enzyme is chemically attached to one of the antibodies. When this enzyme-linked antibody binds to its target, a measurable signal is generated upon adding a specific substrate. The enzyme acts on this substrate, producing a detectable change, commonly a color change. The intensity of this color directly indicates the amount of the target substance, making ELISA effective for both qualitative detection and quantitative measurement.
The ELISA Process
An ELISA begins by coating a plastic plate with a “capture” antibody that specifically binds to the target antigen. After the capture antibody adheres, non-specific binding sites are blocked to prevent false signals.
Next, the biological sample is added to the wells. If the antigen is present, it binds to the immobilized capture antibodies. Unbound components are then washed away. A “detection” antibody, which also recognizes the target antigen at a different binding site, is then introduced. This detection antibody is often linked to an enzyme, or a second enzyme-linked antibody is added that binds to the detection antibody.
After another washing step, a substrate specific to the enzyme is added. The enzyme converts this colorless substrate into a colored product. The intensity of the resulting color, measured using a plate reader, directly corresponds to the amount of antigen originally present in the sample.
Common Applications of ELISA
ELISA is a versatile laboratory technique with widespread applications across various fields, including medicine, food safety, and environmental monitoring.
- In medical diagnostics, it detects infectious diseases by identifying antibodies produced in response to pathogens like viruses or bacteria (e.g., HIV, hepatitis, Lyme disease).
- It also detects autoantibodies, which are antibodies that mistakenly target the body’s own tissues, aiding in the diagnosis of autoimmune diseases.
- ELISA measures hormone levels (e.g., in pregnancy tests for HCG) and screens for cancer markers.
- In food safety, it helps detect allergens like peanuts or milk, as well as contaminants and foodborne pathogens, ensuring product quality and consumer safety.
- Additionally, ELISA is used for drug monitoring, toxicology screening, and in research to quantify proteins and other biomolecules.
Different Types of ELISA
ELISA can be configured in several ways, with four main types: direct, indirect, sandwich, and competitive. Each type varies in its setup, particularly how the antigen and antibodies are immobilized and detected.
In a direct ELISA, the target antigen is coated onto the plate, and a single enzyme-linked antibody binds directly to this antigen. Indirect ELISA uses an unlabeled primary antibody that binds to the antigen, followed by an enzyme-linked secondary antibody. This amplification step can increase sensitivity compared to direct ELISA.
Sandwich ELISA uses a capture antibody to immobilize the antigen, followed by a detection antibody, creating a “sandwich” of antibodies around the antigen. Competitive ELISA measures antigen concentration by observing its competition with a known, labeled antigen for binding to a limited number of antibody sites. A higher concentration of the sample antigen results in a lower signal.