Protein carbonyl measurement by a sensitive ELISA method

Area of Science:

• Biochemistry
• Immunology
• Biomedical Engineering

Background:

• Oxidative injury is implicated in numerous diseases.
• Protein carbonyls are a reliable biomarker of oxidative stress.
• Existing methods for protein carbonyl detection have limitations in sensitivity and sample requirements.

Purpose of the Study:

• To develop and validate a novel Enzyme-Linked Immunosorbent Assay (ELISA) for quantifying protein carbonyls.
• To establish a sensitive and discriminatory assay for oxidative injury assessment.
• To enable analysis of protein carbonyls in limited clinical and experimental samples.

Main Methods:

• Protein samples were derivatized with dinitrophenylhydrazine.
• ELISA plates were used to immobilize derivatized proteins.
• Detection involved a commercial antibody against protein-conjugated dinitrophenylhydrazine, followed by biotin-conjugated antibody and streptavidin-biotinylated horseradish peroxidase.
• Quantification was achieved through spectrophotometric measurement.
• Assay calibration was performed using oxidized albumin.

Main Results:

• The ELISA method demonstrated good correlation with a colorimetric carbonyl assay.
• The assay requires only 60 µg of protein.
• Sensitivity was established in the 0-2.5 nmol/mg protein range, with linearity up to 10 nmol/mg protein.
• The method was successfully applied to analyze protein carbonyls in plasma and lung aspirate samples.

Conclusions:

• The developed ELISA method provides a sensitive and discriminatory assay for protein carbonyls.
• This assay is suitable for analyzing experimental and clinical samples, particularly those with low protein carbonyl concentrations or limited availability.
• The method offers a significant advancement over traditional colorimetric assays for assessing oxidative injury.