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

Area of Science:

• Biochemistry
• Clinical Chemistry
• Analytical Chemistry

Background:

• Conventional d-ROMs and BAP serum tests are valuable oxidative balance markers.
• Current assays require 30 µL serum and 10 minutes per sample, limiting large studies due to cost and throughput.
• Existing methods offer calibration-free operation and flexibility but are not scalable.

Purpose of the Study:

• To develop a high-throughput, cost-effective assay system for d-ROMs and BAP measurements.
• To reduce serum sample volume and labor time for oxidative stress marker analysis.
• To enable large-scale research on oxidative stress and health.

Main Methods:

• A semi-automated system using a pipetting robot and 384-well plates was developed.
• The system processes up to 144 samples (288 assays) simultaneously using only 5.0 µL serum per sample.
• Optimized incubation and sample preparation (centrifugation for BAP) were established for linearity and stability.

Main Results:

• The new method significantly reduced serum requirements (83%) and reagent use (86-93%).
• Labor time decreased by approximately 80% while maintaining high agreement with conventional methods for non-turbid samples.
• The system accurately measured oxidative stress markers even in lipemic and turbid serum samples, with low coefficients of variation (1-17%).

Conclusions:

• A high-throughput, cost-effective in-house assay system for d-ROMs and BAP has been developed.
• This system facilitates accurate oxidative stress marker measurement in large-scale studies, even with challenging sample types.
• The optimized assay supports robust, evidence-based research on oxidative stress and its relation to health.