Enzyme-Linked Immunosorbent Assay (ELISA)

Definition

• ELISA is a widely used immunological technique that detects and quantifies specific molecules, such as proteins, peptides, antibodies, and hormones, in biological samples.

Principle

• ELISA relies on antigen-antibody interactions.

• A specific antigen or antibody is immobilized onto a solid support and subsequently detected using a labeled antibody or enzyme.

Types of ELISA

• Direct ELISA: Uses a single, enzyme-labeled antibody.

• Indirect ELISA: Uses a primary antibody and an enzyme-labeled secondary antibody.

• Sandwich ELISA: Uses two antibodies, one for capturing and the other for detection.

• Competitive ELISA: Measures antigen concentration by competing with a labeled antigen for antibody binding.

Steps of ELISA

1. Coating: The antigen or antibody is immobilized on a solid surface (typically a 96-well plate).

2. Blocking: Unoccupied sites are blocked with a non-specific protein to prevent non-specific binding.

3. Incubation with Primary Antibody: A specific antibody binds to the target antigen.

4. Secondary Antibody Addition: A secondary antibody conjugated with an enzyme (e.g., HRP or alkaline phosphatase) binds to the primary antibody.

5. Substrate Addition: A substrate is added, which reacts with the enzyme to produce a detectable signal (colorimetric or fluorescent).

6. Measurement: The intensity of the color change is measured using a spectrophotometer.

Applications of ELISA

1. Detection of viral and bacterial infections.

2. Detection of autoimmune diseases.

3. Drug discovery and development.

4. Environmental monitoring.