A 1-hour Enzyme-Linked Immunosorbent Assay for Quantitation of Acrolein- And Hydroxynonenal-Modified Proteins by Epitope-Bound Casein Matrix Method

Anal Biochem. 1999 Jun 1;270(2):323-8. doi: 10.1006/abio.1999.4073.

Abstract

A simple and rapid enzyme-linked immunosorbent assay (ELISA) method for quantitation of acrolein and 4-hydroxy-2-nonenal (HNE)-modified proteins was developed. 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 antibody 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 akin to diffusion-rate-limiting reactions. The convenient ELISA should find an application for analyses of the intricate processes involved in oxidative stress and carcinogenic insult. The epitope-attachment methodology may also be advantageous for the quantitation of various other biologically important haptenic molecules.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acrolein
  • Aldehydes
  • Animals
  • Antibodies, Monoclonal
  • Caseins
  • Cattle
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Epitopes
  • Humans
  • Immunochemistry
  • Ovalbumin / analysis
  • Ovalbumin / chemistry
  • Ovalbumin / immunology
  • Proteins / analysis*
  • Proteins / chemistry*
  • Proteins / immunology
  • Rats

Substances

  • Aldehydes
  • Antibodies, Monoclonal
  • Caseins
  • Epitopes
  • Proteins
  • Acrolein
  • Ovalbumin
  • 4-hydroxy-2-nonenal