A Fluorescent Assay for Free Unconjugated Bilirubin

描述（由申请方提供）：目前严重新生儿高胆红素血症的诊断试验与新生儿胆红素脑病相关性不好，导致黄疸婴儿管理的不确定性。为了解决这个问题，将开发一种新的胆红素测定法来测定游离未结合胆红素（UCB）的血清浓度，这是神经系统损伤风险的更好指标。在该项目的第一阶段，将从脂肪酸结合蛋白（FABP）的荧光标记突变体中产生能够检测游离UCB的生理和病理生理水平的UCB特异性探针。该阶段的具体目标是（1）生成与模板探针相比对UCB具有改善亲和力的探针，以及（2）证明这些探针可以测量游离UCB的生理相关浓度，而其他血清组分的干扰可忽略不计。为了实现第一个目标，将使用先前发现的UCB探针作为模板，通过组合诱变FABP结合口袋内的选择残基来产生不结合脂肪酸的荧光标记的FABP突变体的文库。当探针暴露于规定的游离浓度的UCB时，通过定量荧光变化筛选该文库的UCB灵敏度。第二个目标将需要潜在的探针的荧光特性，血清内的潜在干扰源的调查，并从患者的高胆红素血症样本的测量。第二阶段将涉及确定黄疸婴儿的游离UCB水平，将这些结果与结果相关联，并在光疗期间监测游离UCB。

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.