Enzyme-Linked Immunosorbent Assay ( ELISA )

Enzyme-Linked Immunosorbent Assay ( ELISA )

ELISA Technique Overview

The ELISA (Enzyme-Linked Immunosorbent Assay) was conceptualized and developed in 1971 by Swedish scientists Peter Perlmann (principal investigator) and Eva Engvall at the University of Stockholm.In the late 1960s, Stratis Avrameas and G.B. Pierce pioneered immunoenzymology, a method based on the specific reaction between antigens and antibodies, using enzymes as markers to detect and quantify the interaction.

Principle of ELISA

ELISA (Enzyme-Linked Immunosorbent Assay) is an immunoenzymatic detection technique that visualizes the interaction between antigens and antibodies.The principle relies on an enzyme linked to the antibody, which acts on a specific substrate to produce a color change, indicating the presence and quantity of the target antigen.

Indirect ELISA Test:
This assay is used to detect or quantify specific antibodies. It is performed in four steps:

  1. Antigen Coating: The target antigen is immobilized on the surface of the plate.
  2. Primary Antibody Binding: The antibody of interest is added and binds to the coated antigen.
  3. Secondary Antibody Binding: A labeled detection antibody, which recognizes the primary antibody, is added.
  4. Signal Detection: A substrate is introduced to reveal the bound antibodies through a measurable color change.

Indirect ELISA Test – Step-by-Step Procedure

Step 1: Antigen Coating

  • Incubate the wells overnight at 4 °C with the solution containing the specific antigen for the target antibody.
  • Antigen binds to the well surface via electrostatic interactions.
  • Wash the wells with washing buffer to remove any unbound antigen.

Step 2: Primary Antibody Incubation

  • Add the sample containing the antibody to be measured.
  • The antibodies bind specifically to the immobilized antigen.
  • Wash the wells to remove unbound primary antibodies.

Step 3: Detection Antibody Incubation

  • Add the detection antibody, which binds specifically to the primary antibody.
  • Wash the wells to remove excess detection antibody.
  • The detection antibody is conjugated to an enzyme, which converts its substrate into a measurable reaction product, producing a visible color change.

Step 4: Signal Development

  • Incubate the wells at room temperature in the dark with a substrate solution for the enzyme.
  • The appearance of color indicates the presence of the target antibody.
  • The intensity of the color is proportional to the amount of enzyme present, and therefore to the concentration of the antibody in the sample.*

 

ELISA Results Interpretation

  • Qualitative ELISA: Indicates the presence or absence of the antigen in the sample. Threshold values are determined by the analyst and can be based on statistical methods.
  • Quantitative ELISA: Measures the concentration of the antigen. The optical density (OD) or fluorescence units of the sample are interpolated from a calibration (standard) curve to determine the exact amount.

Competitive ELISA Technique

Principle:
The assay uses the target protein immobilized on a plastic surface and a specific enzyme-conjugated antibody, which is revealed by adding a color-producing substrate.
In this competitive system, the enzyme-conjugated antibody competes with the target protein in the sample. If the sample contains the target protein, it binds the conjugated antibody, preventing it from binding to the immobilized protein.
As a result, the more protein present in the sample, the weaker the color signal on the plastic surface this system works inversely compared to standard ELISA.

Advantages:

  • Use of monoclonal antibodies ensures specific detection.
  • Quantification is possible using a standard curve run in parallel.
  • Secondary antibodies increase the sensitivity of the assay.
  • Technique is accessible to all biologists.
  • Signal detection does not require specialized equipment.
  • Commercial kits are typically valid for about one year.

Limitations / Considerations:

  • Detection limit is lower than radioimmunoassays (RIA).
  • Enzymatic reaction is sensitive to temperature, pH and light exposure.

Sandwich ELISA

Principle:
Sandwich ELISA is used to detect specific antigens. In this method, a capture antibody is immobilized on the microtiter plate well. The sample containing the antigen is added and binds to the immobilized antibody, forming an antigen–antibody complex.

Detection:
After washing away unbound materials, a second enzyme-conjugated antibody, which recognizes a different epitope on the antigen, is added. This secondary antibody binds to the captured antigen. Unbound secondary antibody is removed by washing.

Signal Development:
A substrate for the enzyme is added, and the enzyme catalyzes its conversion into a detectable colored product. The intensity of the color is proportional to the antigen concentration in the sample.

Procedure of ELISA Sandwich 

  1. Coating: Prepare the plate by immobilizing a known amount of capture antibody on the well surface.
  2. Antigen Binding: Add the sample containing the antigen and incubate at 37 °C to allow antigen antibody complex formation.
  3. Washing: Wash the wells to remove any unbound antigen.
  4. Detection Antibody: Add enzyme-conjugated detection antibodies specific to a different epitope of the antigen and incubate at 37 °C.
  5. Washing: Wash the wells to remove any unbound detection antibody.
  6. Substrate Reaction: Add the enzyme substrate, which is converted by the enzyme into a colored product. The color intensity is proportional to the antigen concentration in the sample.