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

Abstract

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: 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%.

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.