A Prototype of Ultrasensitive Time-Resolved Fluoroimmunoassay with Enhanced Fluorescence System for the Trace Determination of Urinary 8-Hydroxy-2`-Deoxyguanosine, the DNA Oxidative Stress Biomarker.

This study presents a new highly sensitive and specific time-resolved fluoroimmunoassay (TRFIA) for the measurement of trace amounts of the urinary 8-hydroxy-2`-deoxyguanosine (8-OHdG) which is a biomarker for oxidative stress on DNA. The assay relied on a competitive binding approach and a mouse monoclonal antibody which recognized 8-OHdG with high specificity. In this assay, 8-OHdG conjugated with bovine serum albumin protein (8-OHdG-BSA) was employed as a solid phase antigen. The competition occurred between the 8-OHdG present in the sample solutions and the assay plate-coated 8-OHdG-BSA for a limited quantity of the anti-8-OHdG antibody labeled with a chelate of europium of ethylenediaminetetraacetic acid. The fluorescence signal of the europium chelate-labeled antibody was enhanced by a solution composed of thenoyltrifluoroacetone, trioctylphosphine oxide, and Triton X-100. The validation demonstrated a working range of 10-600 pg mL and a limit of quantitation of 10 pg mL. When applied to urine samples, the assay exhibited satisfactory accuracy and precision in quantifying 8-OHdG. In summary, this study introduces the first TRFIA capable of detecting urinary levels of 8-OHdG at picogram levels. The assay outperforms existing analytical techniques for 8-OHdG in terms of sensitivity, convenience, and analysis throughput.