A Fluorescent Assay for Free Unconjugated Bilirubin-Phase II

描述（由申请人提供）：约60%的新生儿发生黄疸。当严重时，与黄疸相关的胆红素（高胆红素血症）血液水平升高会造成神经损伤的重大风险。目前对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没有很好的相关性，这导致了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）的血浆浓度，这是神经损伤风险的更好指标。在该项目的第一阶段，UCB特异性探针能够检测新生儿血浆中游离UCB（UCBf）的生理和病理生理水平，由脂肪酸结合蛋白（FABP）的荧光标记突变体产生。II期的具体目的是（1）使用800例新生儿的血液样本确认I期UCB探针在新生儿血浆中的准确性，（2）确定光疗期间产生的结合胆红素和胆红素异构体的潜在干扰效应，以及（3）通过改进探针特性和测定形式优化临床使用的测定。为了实现第一个目标，将UCBf的探针测量值与总血清胆红素的临床测量值和UCBf的过氧化物酶测量值进行比较。第二个目标将包括结合胆红素的酶促合成，用于体外和体内光疗处理样品的干扰试验和UCBf测量。第三个目标将需要改进灵敏度、精度和稳定性的新型探头。为了增加灵敏度，将使用I期UCB探针作为模板，通过FABP结合口袋内的选择残基的组合诱变来产生不结合脂肪酸的荧光标记的FABP突变体的新文库。通过向UCB探针中添加第二个荧光团以创建荧光比率探针来提高精度。探针稳定性将通过改变荧光团-FABP缀合化学来增加。第三个目标还将包括优化UCBf测定以用于高通量形式，以促进未来的III期临床试验，以研究UCBf和胆红素神经毒性之间的相关性。这项研究的成功结果将是一种独特的UCBf检测方法，可以更准确地测量新生儿中胆红素介导的神经损伤的风险，并显着改善光疗治疗高胆红素血症的应用。目前胆红素血浆浓度升高的诊断试验与新生儿胆红素脑病相关性不好，这导致黄疸婴儿管理的不确定性。为了解决这一问题，将开发一种新的胆红素测定法来测定游离未结合胆红素的血浆浓度，这是神经损伤风险的更好指标。临床使用该检测试剂盒将通过减少不必要的治疗和提高对胆红素脑病风险婴儿的识别来加强对黄疸婴儿的护理。

DESCRIPTION (provided by applicant): Jaundice occurs in approximately 60% of newborns. When severe, the elevated blood levels of bilirubin (hyperbilirubinemia) associated with jaundice pose a significant risk for neurological damage. Current diagnostic tests for severe neonatal hyperbilirubinemia do not correlate well with bilirubin encephalopathy in newborns, contributing to the uncertainty in the management of jaundiced infants. To address this problem, a new bilirubin assay will be developed to determine the plasma concentration of free unconjugated bilirubin (UCB), which is a better indicator of risk for neurological damage. In Phase I of this project, a UCB-specific probe able to detect physiologic and pathophysiologic levels of free UCB (UCBf) in neonatal plasma was produced from a fluorescently labeled mutant of a fatty acid binding protein (FABP). The specific aims of Phase II are to (1) confirm the accuracy of the Phase I UCB probe in neonatal plasma using blood samples from 800 neonates, (2) determine the effects of potential interference from conjugated bilirubin and bilirubin isomers produced during phototherapy, and (3) optimize the assay for clinical use through improvements in the probe properties and assay format. To accomplish the first aim, probe measurements of UCBf will be compared to clinical measurements of total serum bilirubin and peroxidase measurements of UCBf. The second aim will include the enzymatic synthesis of conjugated bilirubin for interference testing and UCBf measurements of samples treated with in vitro and in vivo phototherapy. The third aim will require new versions of the probe that have improved sensitivity, precision, and stability. To increase sensitivity, new libraries of fluorescently labeled FABP mutants that do not bind fatty acids will be generated by combinatorial mutagenesis of select residues within the FABP binding pocket, using the Phase I UCB probe as a template. Precision will be improved by adding a second fluorophore to the UCB probe to create a fluorescence ratio probe. Probe stability will be increased by altering the fluorophore-FABP conjugation chemistry. The third aim will also include the optimization of the UCBf assay for use in a high throughput format to facilitate future Phase III clinical trials to study the correlation between UCBf and bilirubin neurotoxicity. The successful outcome of this study will be a unique assay for UCBf that more accurately gauges the risk of bilirubin-mediated neurological damage in neonates and significantly improves the application of phototherapy for the treatment of hyperbilirubinemia. Current diagnostic tests for elevated plasma concentrations of bilirubin do not correlate well with bilirubin encephalopathy in newborns, which contributes to the uncertainty in the management of jaundiced infants. To address this problem, a new bilirubin assay will be developed to determine the plasma concentration of free unconjugated bilirubin, which is a better indicator of risk of neurological damage. Clinical use of this assay would enhance the care of jaundiced infants by reducing unnecessary therapy and improving the identification of infants at risk for bilirubin encephalopathy.