Principle of Assay
Tetrahydrobiopterin ELISA Kit (A270605) employs the competitive enzyme immunoassay technique for the quantitative measurement of Tetrahydrobiopterin in serum, plasma, tissue homogenates or other biological fluids. The 96-well microtiter plate has been pre-coated with Tetrahydrobiopterin antigen. During the incubation, Tetrahydrobiopterin present in the samples or standards competes with the fixed amount of immobilized Tetrahydrobiopterin for binding sites on the Biotinylated Anti-Tetrahydrobiopterin Antibody. The more Tetrahydrobiopterin present in a sample or standard, the less Biotinylated Anti-Tetrahydrobiopterin Antibody that binds to the plate. Following incubation, unbound Biotinylated Anti-Tetrahydrobiopterin 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 Tetrahydrobiopterin present in each sample or standard. The concentration of Tetrahydrobiopterin can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.
