Systematic Veterinary Virology

COMPETITIVE ELISA

A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:

• Unlabeled antibody is incubated in the presence of its antigen.
• These bound antibody/antigen complexes are then added to an antigen coated well.
• The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")
• The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme.
• A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.

For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.