Overview
This lecture covers the principles and steps of the ELISA (Enzyme-Linked Immunosorbent Assay) technique, its types, and an example of how it is used to diagnose HIV infection.
What is ELISA?
- ELISA stands for Enzyme-Linked Immunosorbent Assay.
- The technique uses antibodies to detect the presence and amount of a specific antigen or antibody in a sample.
- An enzyme is attached to an antibody, and a color change upon substrate reaction indicates a positive result.
Types of ELISA
- Direct ELISA uses a single antibody complementary to the antigen being tested.
- Indirect ELISA uses two antibodies: a primary antibody that binds the antigen, and a secondary antibody with an enzyme attached.
Example: Indirect ELISA for HIV Diagnosis
- HIV antigens are fixed to the bottom of a test well.
- The patient’s blood plasma is added; if HIV-specific antibodies (primary) are present, they bind to the antigens.
- The well is washed to remove unbound antibodies.
- Secondary antibodies with enzymes are added, binding to any primary antibodies present.
- Another wash removes unbound secondary antibodies, reducing false positives.
- A substrate is added; if the enzyme is present, a color change occurs.
- A color change indicates the presence of HIV antibodies, confirming HIV infection.
Key Terms & Definitions
- ELISA — A laboratory test to detect and measure antigens or antibodies using enzyme-linked antibodies.
- Antigen — A substance that induces an immune response, often a protein on a pathogen.
- Antibody — A protein produced by B-cells that specifically binds to an antigen.
- Substrate — A molecule on which an enzyme acts, causing a detectable signal (color change).
- Primary Antibody — The first antibody that binds directly to the target antigen.
- Secondary Antibody — Binds to the primary antibody and typically has an enzyme attached.
Action Items / Next Steps
- Review the steps of both direct and indirect ELISA.
- Practice drawing or interpreting flow diagrams for ELISA procedures.
- Prepare for exam questions on the use of ELISA for disease diagnosis, especially HIV.