Protein carbonyl measurement by a sensitive ELISA method

Abstract

We describe a new immunoassay for measuring protein carbonyls as an index of oxidative injury. Protein samples were reacted with dinitrophenylhydrazine then adsorbed to wells of an ELISA plate before probing with a commercial antibody raised against protein-conjugated dinitrophenylhydrazine. The biotin-conjugated primary antibody was then reacted with streptavidin-biotinylated horseradish peroxidase for quantification. The method was calibrated using oxidized albumin and results correlated well with the colorimetric carbonyl assay. The method required only 60 microg protein and was used to analyze the amount of protein carbonyls in plasma and lung aspirate samples. It was sensitive in the 0-2.5 nmol/mg protein range within which clinical samples fell and was linear up to 10 nmol/mg protein. The ELISA method for protein carbonyls is more sensitive and discriminatory than the colorimetric assay and should have wide application for analysing experimental and clinical samples, especially where concentrations are low and where only small amounts of sample are available.