Enzyme-Linked Immunosorbent Assay (ELISA): Principle and Procedure

Introduction to ELISA: The Enzyme-Linked Immunosorbent Assay (ELISA) is a widely used laboratory technique for detecting and quantifying specific proteins, antibodies, antigens, or hormones in biological samples. Its high sensitivity and specificity make it a standard tool in diagnostics, immunology, and biotechnology research.

Principle of ELISA: ELISA operates on the basic principle of antigen-antibody binding. The target molecule (antigen or antibody) is immobilized on a solid surface, typically a microtiter plate. This immobilized target is then exposed to a specific antibody that binds to it. An enzyme is linked to this antibody, and upon adding a substrate, a detectable color change occurs due to the enzyme’s action on the substrate. The intensity of the color is proportional to the amount of the target molecule present in the sample.

Types of ELISA:

1. Direct ELISA: Involves directly labeling the primary antibody with an enzyme.
2. Indirect ELISA: Uses a secondary antibody with an enzyme label to detect the primary antibody.
3. Sandwich ELISA: Utilizes two antibodies, where the antigen is ‘sandwiched’ between the capture and detection antibodies.
4. Competitive ELISA: Involves competition between the sample antigen and a labeled antigen for binding to the antibody.

ELISA Procedure:

1. Coating: The microtiter plate wells are coated with an antigen (or antibody) specific to the analyte being measured.
2. Blocking: Non-specific binding sites are blocked using a blocking agent, like bovine serum albumin (BSA).
3. Sample Addition: The sample containing the target antigen (or antibody) is added to the wells, where it binds to the coated antibody (or antigen).
4. Primary Antibody Addition: In indirect and sandwich ELISA, a primary antibody specific to the antigen is added, followed by washing to remove unbound substances.
5. Enzyme-Linked Antibody: An enzyme-linked secondary antibody is added, binding to the primary antibody.
6. Substrate Addition: A substrate (like TMB) is added, which reacts with the enzyme to produce a color change.
7. Detection: The color change is measured using a spectrophotometer. The intensity of the color correlates with the concentration of the target molecule.

Applications of ELISA:

• Detection of infectious diseases (HIV, hepatitis).
• Measurement of hormones, cytokines, and other biomolecules.
• Monitoring immune responses in vaccine development.

ELISA remains a cornerstone in clinical and research laboratories due to its precision, scalability, and ease of use.