Human High Mobility Group Box1 Protein ELISA Kit

The coated well immunoenzymatic assay for the quantitative measurement of serum HMGB1 utilizes a monoclonal anti-HMGB1 and a HMGB1-HRP conjugate. The assay asample and buffer are incubated together with anti-HMGB1 antibody coated plate for sixty and washed. The diluted HMGB1-HRP conjugate is then added to each well and incubated. After the incubation period, the wells are decanted and washed three times. The wells are then incubated with a substrate for the enzyme. The product of the enzyme-substrate reaction forms a blue colored complex. Finally, a stopping 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.Standards of known HMGB1 concentrations are run concurrently with the samples being assayed and a standard curve is plotted relating the intensity of the color (Optical Density) to the concentration of HMGB1. The unknown HMGB concentration in each sample is interpolated from this curve.