Principle of Assay
Human Sphingosine-1-phosphate ELISA Kit (A73746) employs the competitive enzyme immunoassay technique for the quantitative measurement of human Sphingosine-1-phosphate in serum, plasma, tissue homogenates, and other biological fluids. The 96-well microtiter plate has been pre-coated with Sphingosine-1-phosphate antigen. During the incubation, Sphingosine-1-phosphate present in the samples or standards competes with the fixed amount of immobilized Sphingosine-1-phosphate for binding sites on the Biotinylated Anti-Sphingosine-1-phosphate Antibody. The more Sphingosine-1-phosphate present in a sample or standard, the less Biotinylated Anti-Sphingosine-1-phosphate Antibody that binds to the plate. Following incubation, unbound Biotinylated Anti-Sphingosine-1-phosphate Antibody is removed by washing, and an HRP-Streptavidin 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 Sphingosine-1-phosphate present in each sample or standard. The concentration of Sphingosine-1-phosphate can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.
