Description
Principle of the Assay: HMGB1 ELISA kit applies the competitive enzyme immunoassay technique utilizing a monoclonal anti-HMGB1 antibody and an HMGB1-HRP conjugate. The assay sample and buffer are incubated together with HMGB1-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 HMGB1 concentration since HMGB1 from samples and HMGB1-HRP conjugate compete for the anti-HMGB1 antibody binding site. Since the number of sites is limited, as more sites are occupied by HMGB1 from the sample, fewer sites are left to bind HMGB1-HRP conjugate. A standard curve is plotted relating the intensity of the color (O.D.) to the concentration of standards. The HMGB1 concentration in each sample is interpolated from this standard curve