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

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.

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).

Based on nonlinearity testing, 450 nm was selected as the optimal wavelength. To maintain linearity across various sample types, including turbid serum, d-ROMs required incubation at 4 °C for one week, and BAP required centrifugation to remove precipitates. Intra- and inter-assay, as well as inter-day, coefficients of variation ranged from 1% to 9% for d-ROMs and 2% to 17% for BAP. Inter-operator variations were <9%. Lipemic serum showed abnormally low BAP values using the conventional method but normal values using our method. In non-turbid samples, the current and conventional methods showed high agreement. Deming regression analysis of log₁₀-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%.

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.