Abstract
Otago BMLSc student research project abstract.
Objectives: Oxidative stress is associated with various diseases and ageing. Physiological oxidants can directly oxidise proteins, forming stable carbonyl groups. Protein carbonyls are a general biomarker of oxidative stress and are traditionally quantified by derivatisation with 2,4-dinitrophenylhydrazine (DNP), followed by detection with an antibody. Our laboratory recently redeveloped an ELISA assay that replaced DNP and a biotinylated anti-DNP antibody with biotin hydrazide (BH), removing the requirement for an antibody. This study aimed to test and validate various aspects of the new assay.
Methods: In this ELISA-based assay, protein samples are derivatised with BH and bound to a plate. Streptavidin- horseradish peroxidase binds strongly to the biotin group, and tetramethylbenzidine oxidation is used to detect and measure protein carbonyls. Critical steps in the protocol were slightly modified and tested to determine the impact on protein carbonyl quantification.
Results: Comparable performance was observed between freshly reconstituted standards and those subsequently stored at 4°C for one week. The optimum BH concentration for derivatisation was found to be 2 mM (tested range: 1-4 mM). No statistically significant differences were observed between derivatisation time variations (30-90 minutes). No significant difference was observed when the amount of protein added to wells was varied (0.5-2.0 ug). Varying chromogen development time did not affect protein carbonyl levels. The assay was determined to have a limit of detection of 0.08 nmol carbonyls/ mg protein.
Conclusion: Critical evaluation of various parameters in the new assay confirmed its robustness to slight deviations from the protocol and identified optimal conditions.