Fluorescent protein-based detection of unconjugated bilirubin in newborn serum

Kernicterus or bilirubin-induced neurologic dysfunction is a brain disorder caused by bilirubin neurotoxicity during the neonatal period1. Not only does this disease show worldwide prevalence, its incidence is increasing in developed countries due to the higher survival rates of extremely preterm infants2,3. Although assessment of total bilirubin (TB) levels in serum/plasma is the current gold standard for identifying newborns at risk of kernicterus4,5, TB level is not the most accurate indicator, because TB includes both unconjugated bilirubin and conjugated bilirubin. Of these fractions, elevated serum level of unconjugated bilirubin in newborns is associated with increased risk of developing kernicterus6,7. Conventional methods have limited applicability since unconjugated bilirubin is insoluble in water at physiologic pH and temperature8,9; in current clinical settings, serum levels of unconjugated bilirubin are indirectly determined as the difference between total and conjugated bilirubin levels, which are measured with the diazo method, bilirubin oxidase method, or by spectrophotometry10. A major disadvantage of these conventional methods is to produce an inaccurate result in serum samples with turbidity. For example, the bilirubin oxidase method measures bilirubin levels by monitoring a change in absorbance at 450 nm, which is affected by turbidity due to hemoglobin or lipid emulsion11. High-performance liquid chromatography is the only method for directly measuring unconjugated bilirubin12, but is impractical for routine clinical use. The recently cloned UnaG, fluorescent protein from eel muscle, specifically binds to the unconjugated but not the conjugated form of bilirubin with high affinity13. In this study, we used UnaG to directly measure unconjugated bilirubin levels in newborn serum or whole blood. Our findings demonstrate that the UnaG method is highly specific and sensitive and can be useful in a clinical setting.