| Background | Protein A ELISA kit applies the quantitative sandwich enzyme immunoassay technique. The microtiter plate has been pre-coated with a monoclonal antibody specific for Protein A. Samples containing Protein A are first diluted in the Sample Diluent provided with the kits. The Denaturing Buffer is then added and mixed to dissociate the Protein A from the product antibody. The samples are then reacted in microtiter strips coated with a monoclonal anti-Protein A capture antibody. A second anti-Protein A biotinylated monoclonal antibody is simultaneously reacted forming a sandwich complex of solid phase antibody-Protein A: biotin labeled antibody. After a wash step to remove any unbound reactants, Streptavidin labeled HRP is added to each well. After a short incubation period, the strips are then reacted with TMB substrate. The enzyme-substrate reaction is terminated by addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. A standard curve is plotted relating the intensity of the color (OD) to the concentration of standards. The Protein A concentration in each sample is interpolated from this standard curve. |
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