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Principle of ELISA (Enzyme-linked Immunosorbent Assay) Test

Identify Antibody or Antibody Pairs

Accurately measuring the binding of antibody with antigen by an ELISA will depend on the specificity of the antibody for the antigen. Poor specificity of the antibody will result in highly nonspecific background. In contrast, specific but weak binding may be washed away, resulting in a falsely low signal. Choosing proper antibodies must thus be performed in order to avoid these issues as well as crossreactivity between antibodies within the assay.

A sandwich ELISA requires two antibodies: one used for capture and the other for detection. They are often referred to as a “matched pair.” In most cases, sandwich assays are more specific because the assay requires 2 antibodies to recognize the target of interest.

CST offers ELISA kits where optimal antibody pairs have been identified, delivering robust and reproducible data for each experiment.

Coating ELISA Plate

The ELISA plate is coated with a specific antibody or antigen using the appropriate buffer. Conditions need to be optimized for passive adsorption, as they can be influenced by several factors, including surface chemistry of the plastic, temperature, pH of the coating buffer, antigen/antibody concentration, and time.

Typical coating buffers include phosphate-buffered saline (PBS), sodium bicarbonate, or similar buffers, but these conditions should be tested and optimized. Importantly, coating buffers should not contain proteins that can compete with the binding of the antigen or antibody.

Capturing Antigen with Antibodies

The method for antigen capture will depend on the ELISA type selected:

  • Direct ELISA - Enzyme conjugated antibody binds to antigen on a surface.
  • Indirect ELISA - Similar to direct ELISA, but primary binding antibody is not conjugated. A second conjugated antibody is used to detect the primary antibody.
  • Sandwich ELISA - The antigen is recognized by 2 antibodies specific for different epitopes, making a complex like a sandwich.


Detection of Analytes

After the antibody detection of the analyte, the substrate is added to the well. The antibody enzyme conjugate reacts with the substrate to produce a colorimetric, chemiluminescent, or fluorescent signal. The resulting signal is typically measured on a microplate reader.

Incubation time and temperature can be modified to give optimal signal to noise ratio.