Protein Carbonyl Measurement by ELISA

Abstract

Protein carbonyls are formed by a variety of oxidative mechanisms and are sensitive indices of oxidative injury (1). The conventional assay for protein carbonyls is a colorimetric procedure that measures binding of dinitrophenylhydrazine (DNP) (2,3). Protein-bound DNP can also be measured with increased sensitivity by enzyme-linked immunosorbent assay (ELISA) (4,5). 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 of serum albumin containing increasing proportions of hypochlorous acid (HOCl)- oxidized protein that is calibrated colorimetrically. This method requires only microgram quantities of protein and avoids the high and sometimes variable blanks owing to unbound DNP that are limitations for the colorimetric method (6,7). We have found it to be highly sensitive and discriminatory in analyzing plasma and lung aspirates from both critically ill adult patients and premature infants (8,10), and others have used it for cell extracts (11). Results correlate well with the colorimetric assay. Absolute values can differ between the two assays and each method is best suited for comparing samples using a standard procedure.