Principle of Assay
Canine Insulin ELISA Kit (A82295) employs the competitive enzyme immunoassay technique for the quantitative measurement of canine Insulin in serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids. The 96-well microtiter plate has been pre-coated with Insulin antigen. During the incubation, Insulin present in the samples or standards competes with the fixed amount of immobilized Insulin for binding sites on the Biotinylated Anti-Insulin Antibody. The more Insulin present in a sample or standard, the less Biotinylated Anti-Insulin Antibody that binds to the plate. Following incubation, unbound Biotinylated Anti-Insulin Antibody is removed by washing, and an HRP-Avidin conjugate is added to the wells and the microtiter plate is incubated. Following incubation and washing, TMB substrate solution is then used to visualize the HRP enzymatic reaction by catalysis to produce a blue-coloured product that changes to yellow after addition of acidic stop solution. The density of yellow is inversely proportional to the amount of Insulin present in each sample or standard. The concentration of Insulin can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.
