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

ENZYME IMMUNE ASSAYS(EIAS)

• EIAs, commonly called as ELISAs (enzyme linked immuno sorbant assays) are similar in principle to RIA but depend on an enzyme rather than a radio active label.
• An enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product. Such a substrate is called a chromogenic substrate.
• A number of enzymes have been employed for ELISA, including alkaline phosphatase, horse radish peroxidase and β-galactosidase. These assays match the sensitivity of RIA and have advantage of being safer and less costly.
• The various chromogenic substrates used are PNPP (para nitrophenyl phosphate) for alkaline phosphatase (conjugate), ONPG (O-nitro phenyl β – D – galactopyranoside) for β- galactosidase and OPD (ortho phenylene diamine), TMB (tetra methyl benzidine), ABTS (2,2’-Azino diethylbenzothiazoline sulfonic acid), 5 AS (5 - Amino salicylic acid) and DAB (diamino benzidine) for horse radish peroxidase. DAB forms an insoluble product.
• Different forms of ELISA are
  • Direct ELISA
  • Indirect ELISA
  • Sandwich ELISA
  • Competitive ELISA
  • Avidin biotin ELISA
  • Dot ELISA
  • Dipstick ELISA