A high-throughput biliverdin assay using infrared fluorescence

Biliverdin is an intermediate of heme degradation with an established role in veterinary clinical diagnostics of liver-related diseases. The need for chromatographic assays has so far prevented its wider use in diagnostic laboratories. The current report describes a simple, fast, high-throughput, and inexpensive assay, based on the interaction of biliverdin with infrared fluorescent protein (iRFP) that yields functional protein exhibiting infrared fluorescence. The assay is linear in the range of 0-10 mu mol/l of biliverdin, has a limit of detection of 0.02 mu mol/l, and has a limit of quantification of 0.03 mu mol/l. The assay is accurate with relative error less than 0.15, and precise, with coefficient of variation less than 5% in the concentration range of 2-9 mu mol/l of biliverdin. More than 95% of biliverdin was recovered from biological samples by simple dimethyl sulfoxide extraction. There was almost no interference by hemin, although bilirubin caused an increase in the biliverdin concentration, probably due to spontaneous oxidation of bilirubin to biliverdin. The newly developed biliverdin assay is appropriate for reliable quantification of large numbers of samples in veterinary medicine.