A Fluorescent Assay for Free Unconjugated Bilirubin

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DESCRIPTION (provided by applicant): Current diagnostic tests for severe neonatal hyperbilirubinemia do not correlate well with bilirubinencephalopathy in newboms, contributing to uncertainty in the management of jaundiced infants. To address this problem, a new bilirubin assay will be developed to determine the serum concentration of free unconjugated bilirubin (UCB), which is a better indicator of risk of neurologic damage. In Phase I of this project, a UCB-specific probe able to detect physiologic and pathophysiologic levels of free UCB will be produced from a fluorescentiy labeled mutant of a fatty acid binding protein (FABP). The specific aims of this phase are (1) generate probes with improved affinity for UCB as compared to a template probe and (2) demonstrate that these probes can measure physiologically relevant concentrations of free UCB with negligible interference from other serum components. To accomplish the first aim, a library of fluorescentiylabeled FABP mutants that do not bind fatty acids will be generated by combinatorial mutagenesis of select residues within the FABP binding pocket, using a previously discovered UCB-probe as a template. This library will be screened for UCB sensitivity by quantification of the change in fluorescence when the probesare exposed to defined free concentrations of UCB. The second aim will require the fluorescence characterization of potential probes, the investigation of potential sources of interference within serum, and measurement of hyperbilirubinemic samples from patients. Phase II will involve determining free UCB levels for jaundiced infants, correlating these results to outcome, and monitoring free UCB during phototherapy.