A high-throughput biliverdin assay using infrared fluorescence.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Veterinary Medicine

Background:

• Biliverdin, a heme degradation intermediate, is crucial for diagnosing liver-related diseases in veterinary medicine.
• Current diagnostic methods for biliverdin often require complex chromatographic assays, limiting their widespread use.
• There is a need for simpler, faster, and more accessible biliverdin quantification techniques.

Purpose of the Study:

• To develop and validate a novel, high-throughput assay for biliverdin quantification.
• To utilize the interaction between biliverdin and infrared fluorescent protein (iRFP) for fluorescence-based detection.
• To establish the assay's performance characteristics, including linearity, sensitivity, accuracy, and precision.

Main Methods:

• A new assay was developed based on the interaction of biliverdin with infrared fluorescent protein (iRFP).
• The assay measures the infrared fluorescence emitted by the functional protein complex.
• Dimethyl sulfoxide (DMSO) was used for sample extraction, and interference from hemin and bilirubin was assessed.

Main Results:

• The assay demonstrated linearity in the 0-10 µmol/l biliverdin range, with a limit of detection of 0.02 µmol/l and limit of quantification of 0.03 µmol/l.
• High accuracy (relative error < 0.15) and precision (coefficient of variation < 5%) were achieved.
• Over 95% biliverdin recovery was obtained from biological samples via DMSO extraction, with minimal hemin interference.

Conclusions:

• The newly developed iRFP-based assay provides a simple, fast, and inexpensive method for biliverdin quantification.
• This assay is suitable for high-throughput analysis in veterinary clinical diagnostics.
• The assay overcomes previous limitations, enabling broader application in diagnosing liver-related diseases.