Abstract
Increased serum levels of unconjugated bilirubin are associated with the development of brain damage in newborns. In current clinical settings, there are no methods for directly determining serum levels of unconjugated bilirubin. UnaG, a fluorescent protein from Japanese eel muscle that specifically binds to unconjugated bilirubin was used in this study. Linear regression analysis was carried out to compare unconjugated bilirubin levels measured by UnaG and conventional bilirubin oxidase methods. Unconjugated bilirubin levels in the serum of newborns who were untreated or treated with phototherapy were compared. Effects of interfering factors in the serum (conjugated bilirubin, hemoglobin, and lipid) on unconjugated bilirubin concentration measured by the UnaG method were also evaluated. Unconjugated bilirubin levels measured by the UnaG method were highly correlated with those determined by the bilirubin oxidase assay. Unconjugated bilirubin levels determined by bilirubin oxidase and UnaG assays were similar in serum samples containing conjugated bilirubin. The performance of the UnaG assay was unaffected by phototherapy and the presence of serum hemoglobin and lipid emulsion. These results demonstrate the clinical applicability of the UnaG method for direct measurement of unconjugated bilirubin levels in newborn serum.
Highlights
With serum concentrations obtained by the bilirubin oxidase and UnaG methods (y = 0.76x + 0.30, r = 0.962 and y = 0.73x + 0.77, r = 0.962, respectively; P < 0.001; Fig. 4A,B); the slopes were not equal to 1 in either case. This is the first large-scale clinical study demonstrating the applicability of eel-derived fluorescent protein for measurement of unconjugated bilirubin concentrations in newborn serum samples
The concentrations measured by this method showed high correlation with those obtained by the conventional bilirubin oxidase method, which has itself shown good correlation with those obtained by high-performance liquid chromatography, considered as the gold standard[12,14,15]
The precision of the method was confirmed by intra- and inter-day assays, which is an important factor for the clinical applicability of this method. These results indicate that the UnaG assay is robust and can be used for direct measurement of unconjugated bilirubin in newborns
Summary
Correlation between unconjugated bilirubin concentrations in serum obtained by bilirubin oxidase and UnaG methods. Unconjugated bilirubin concentrations were similar across 14 serum samples with conjugated bilirubin concentration ≥1 .0 mg/dl as determined by the UnaG and bilirubin oxidase methods (Table 1).
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