High-throughput, low volume d-ROMs and BAP assays: 384-well plate method for large-scale studies

Résumé fourni par la source

BACKGROUND: The d-ROMs and BAP serum tests are promising markers of oxidative balance in research settings. Conventional assay systems require 30 µL of serum and approximately 10 minutes to analyze a single sample. Although these systems have the advantages of calibration-free operation and the flexibility to perform individual tests at any time, their limited throughput and relatively high cost restrict their use in large-scale studies. METHODS: To address this issue, we developed a small-scale, semi-automated measurement system incorporating a pipetting robot and a 384-well plate. The system requires only 5.0 µL of serum sample and can process up to 144 samples simultaneously for both d-ROMs and BAP assays (a total of 288 measurements). RESULTS: -transformed d-ROMs values yielded a slope of 1.06 (95% confidence interval between 0.99-1.13), and an intercept of -0.12 (-0.28 to 0.04). The same analysis for BAP yielded a slope of 0.97 (0.821-1.13) and an intercept of -0.12 (-0.391 to 0.625). Stability testing indicated that serum should be stored at -80 °C in volumes ≥100 µL for standard 1.5 mL tubes or ≥21 µL for 96-well format tubes. Our method reduced serum requirements by 83%, reagent use by 93% for d-ROMs and 86% for BAP. Although colorimetric measurement is required after one week of incubation, actual labor time was reduced by approximately 80%. CONCLUSIONS: We have developed a high-throughput, cost-effective in-house assay system for large-scale studies. This system provides accurate measurements of oxidative stress markers even in turbid samples, facilitating robust evidence-based research on oxidative stress and health.

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