Protein carbonyl measurement by enzyme-linked immunosorbent assay

This chapter discusses the measurement of protein carbonyl by enzyme-linked immunosorbent assay. Protein carbonyls are formed by a variety of oxidative mechanisms and are sensitive indices of oxidative injury. The conventional assay for protein carbonyls is a colorimetric procedure that measures binding of dinitrophenylhydrazine (DNP). Protein-bound DNP can also be measured, with increased sensitivity, either by HPLC (high-performance liquid chromatography), or using an anti-DNP antibody with Western blots or tissue sections. Samples containing protein are reacted with DNP then the protein is nonspecifically adsorbed to an ELISA plate. Unconjugated DNP and nonprotein constituents are easily washed away and give minimal interference. The adsorbed protein is probed with a commercial biotinylated anti-DNP antibody followed by streptavidin-linked horseradish peroxidase. Absorbances are related to a standard curve prepared for bovine serum albumin (BSA) containing increasing proportions of hypochlorous acid 1(HOCl)-oxidized protein that is calibrated colorimetrically.