A 1-hour enzyme-linked immunosorbent assay for quantitation of acrolein- and hydroxynonenal-modified proteins by epitope-bound casein matrix method

A simple and rapid enzyme-linked immunosorbent assay (ELISA) method for qua ntitation of acrolein and 4-hydroxy-2-nonenal (HNE)-modified proteins was d eveloped. Microtiter plate wells were precoated and blocked simultaneously with epitope-bound bovine caseins as matrix proteins, and aldehyde-modified proteins were quantitated by a competition assay with a monoclonal antibod y specific for acrolein-modified lysine or HNE-modified histidine epitopes, Minimal reaction times required for the coating/blocking; first monoclonal antibody and the peroxidase-conjugated second antibody binding steps were 3, 3, and 7 min, respectively, the former two steps being found to be or ak in to diffusion-rate-limiting reactions. The convenient ELISA should find a n application for analyses of the intricate processes involved in oxidative stress and carcinogenic insult. The epitope-attachment methodology may als o be advantageous for the quantitation of various other biologically import ant haptenic molecules. (C) 1999 Academic Press.