VMC 221: Veterinary Immunology and Serology (1+1)

COMPETITIVE ELISA

• Competition ELISA imply that two reactants are trying to bind to a third.
• Proper competition assays involve the simultaneous addition of the two competitors.
• Competitive ELISA for detection of antibody
  • This is essentially the same as the indirect ELISA, except that a competing antibody (eg: antibody in serum to be tested is added to the solid phase antigen either before or simultaneously with pretitrated specific antibody (eg: mouse monoclonal antibody).
  • The competing antibody must be from a different species from the pretitrated antibody since the antispecies conjugate must not react with both. If the competing antibody i.e. antibody in the test serum binds to antigen, it prevents the pretitrated antibody from binding to the antigen.
  • Then the antispecies conjugate directed against the pretitrated specific antibody is added.
  • Then after incubation and washing, the substrate is added and color reaction product is measured.
  • But in this test, presence of antibody in the test serum is denoted by decreased color production because this antibody prevents binding of the pretitrated antibody and we are using antispecies conjugate which detects only bound pretitrated antibodies.