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

COMPONENTS OF INDIRECT ELISA

• Adsorption (coating) of antigen into an ELISA plate and washing off the excess unattched antigen.
• Blocking of unabsorbed areas with bovine serum albumin (BSA).
• Addition of serum, followed by incubation and then washing off the excess unattached antibody.
• Addition of enzyme labeled anti immunoglobulin conjugate, again followed by incubation and washing.
• Addition of enzyme – specific soluble substrate which produces a colour change. This is measured spectrophotometrically.
• In most cases, the enzyme used is horse radish peroxidase. Several soluble substrates have been used for peroxidase enzyme {(Ortho phenylene diamine dihydrochloride (OPD), or Tetra methyl benzidine (TMB), or 2, 2’- azino di (3-ethylbenzothiaoline-6-sulfonic acid) ABTS)}. Different substrates give different colors after enzyme reacation.