Description
Principle of the assay: C5a ELISA kit applies the competitive enzyme immunoassay technique utilizing a monoclonal anti-C5a antibody and an C5a-HRP conjugate. The assay sample and buffer are incubated together with C5a-HRP conjugate in pre-coated plate for one hour. After the incubation period, the wells are decanted and washed five times. The wells are then incubated with a substrate for HRP enzyme. The product of the enzyme-substrate reaction forms a blue colored complex. Finally, a stop solution is added to stop the reaction, which will then turn the solution yellow. The intensity of color is measured spectrophotometrically at 450nm in a microplate reader. The intensity of the color is inversely proportional to the C5a concentration since C5a from samples and C5a-HRP conjugate compete for the anti-C5a antibody binding site. Since the number of sites is limited, as more sites are occupied by C5a from the sample, fewer sites are left to bind C5a-HRP conjugate. A standard curve is plotted relating the intensity of the color (O.D.) to the concentration of standards. The C5a concentration in each sample is interpolated from this standard curve