High-Throughput Measurement of Oxidative Stress Indicators: d-ROMs and BAP Assays in a 384-well Plate Format

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Abstract The body’s oxidative balance is regulated by systems that produce and eliminate reactive oxygen species (ROS), and disruptions in this balance can contribute to the development of lifestyle-related diseases. d-ROMs and BAP tests, which evaluate oxidative balance using just 30 µL of serum per sample in approximately 10 minutes, are widely used for this purpose. However, these methods are not suitable for large-scale studies due to their low-throughput and cost. To overcome these limitations, we established a simultaneous measurement system for multiple samples (144 samples) for measuring d-ROMs and BAP using a pipetting robot and a 384-well plate. The developed system demonstrated good linearity and reproducibility, while significantly reducing reagent and sample consumption. Measurement differences compared to the one of the currently available FREE Carrio Duo system ranged from −5% to 3% for d-ROMs and −12% to 8% for BAP, indicating high consistency with existing methods. Furthermore, the measurement time was substantially shortened from 24 hours (10 minutes × 144 samples) to at longest four hours, although the final color measurements for d-ROMs were performed one week later. This optimized semi-automated system enables the precise and efficient measurement of oxidative stress markers, making it suitable for large-scale studies.

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