ELISA (Enzyme linked Immunosorbent Assay )

 ELISA

Ø  Enzyme-Linked Immunosorbent Assay (ELISA) is a modern molecular technique for the detection of antigen-antibody interaction with the help of an enzyme.

Ø                                     This immunoassay technique relies on antigen-antibody interaction.

Ø  The ELISA test enables the diagnosis of diseases such as HIV, dengue, and COVID-19 by measuring molecules in blood or other body fluids.

Principle of ELISA Technique

Ø  The ELISA technique is based on the specific binding between an antigen and its corresponding antibody. 

Ø  In ELISA, either the antigen or antibody is attached to a solid surface (such as the wells in a microtitre plate).

Ø  An enzyme-linked antibody and antigen is then added. If the target molecule is present in the sample, the antibody-antigen reaction occurs, forming a complex. 

Ø  After adding a substrate specific for the enzyme, a detectable color change is produced, indicating the presence of specific antibody and antigen.

Types of ELISA

1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA

Direct ELISA

Ø  In this method, the antigen is directly detected by a single enzyme-linked antibody.

Ø  Because it uses only one antibody, it is quick, but less sensitive compared to other types.

Steps Of Direct ELISA

1.     Coating the plate

Ø  A 96-well microtiter plate is used (plastic wells).

Ø  Antigen (the target molecule) is added into the wells.

Ø  Antigens stick to the plastic surface by simple adsorption.

2.     Blocking

Ø  After coating, not all surface of the well is covered.

Ø  To prevent unwanted binding, a blocking agent (like BSA – bovine serum albumin, or milk proteins) is added.

Ø  This covers the empty spaces on the plate.

3.     Addition of Enzyme-Linked Primary Antibody

Ø  A primary antibody that specifically binds to the antigen is added.

Ø  This antibody is directly linked with an enzyme (like horseradish peroxidase, HRP, or alkaline phosphatase).

Ø  if antigen is present, the antibody binds directly.

4.     Washing

Ø  Extra unbound antibodies are washed away with buffer solution.

5.     Substrate Addition

Ø  A colorless substrate (chromogenic reagent) is added.

Ø  The enzyme on the antibody reacts with the substrate → produces colored product.

 Example: HRP reacts with TMB (Tetramethylbenzidine) → gives blue color.

References

1. Enzyme-linked immunosorbent assay (ELISA) methodology and principle. Wikipedia. https://en.wikipedia.org/wiki/ELISA

2. Aydin S., Emre E., Ugur K., et al., An overview of ELISA: a review and update on best laboratory practices, Journal of International Medical Research, 2025.

3. ELISA technique, principle & procedure explained. Vedantu, 2025.

4. Thermo Fisher Scientific – ELISA assay technique overview.

5. ELISA method based on antigen-antibody binding. MicrobeNotes, 2022.