Principle of the Procedure        

This kit uses the Sandwich-ELISA principle. The microtiter plate strips has been pre-coated with an affinity purified antibody to Rat IRF4. Standards or samples containing Rat IRF4 are added to the plate and reacted with capture antibody. A second anti-Rat IRF4 antibody labeled biotin is then added and binds to Rat IRF4 captured on the plate. After that, Streptavidin-Horseradish Peroxidase(SA-HRP) is added to form a sandwich complex of solid phase antibody-Rat IRF4-biotin labeled antibody-SA-HRP. And then, TMB substrate solution is added to all wells and incubated. An enzyme-catalyzed reaction generates a blue color in the solution, thereafter, stop solution is added to stop the substrate reaction and the color turns yellow. The yellow solution is read at a wavelength of 450nm. The concentration of Rat IRF4 in the samples is then calculated from the OD value by establishing a standard curve.