NOVEL ENZYMATIC ASSAYS FOR TOTAL AND CONJUGATED BILIRUBI

总胆红素和结合胆红素的新型酶法测定

• 批准号: 2866850
• 负责人: Mary Gaunt-Kloepfer
• 金额: $ 38.84万
• 依托单位: MICRONIX, INC.
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2866850
• 关键词: bilirubin; bioassay; chemical conjugate; enzyme mechanism; fiber optics; high performance liquid chromatography; iron cyanide; method development; oxidoreductase; spectrometry; technology /technique development

This company has proposed the development of novel enzymatic specific assay methods for total, "direct," and conjugated bilirubin. The enzyme bilirubin oxidase (bilrubin, oxido-reductase, EC.1.3.3.5) from myrothecium verrucaria oxidizes bilirubin to bilverdin and higher oxidation products. The reaction is monitored by measuring the decrease in OD460 due to disappearance of endogenous bilirubin. Presence of recyclable ferricyanide in the reagents accelerates and linearizes signal progression and enhances reagent stability. This reduces the amount of enzyme need and facilitates adaptability to analytical equipment. During Phase I we have demonstrated good specificity of the reagents for their targeted bilirubin fractions. Prototype methods were developed for: 1) analysis of diluted serum specimens on centralized clinical analyzer; 2) assay of undiluted micro-specimens at the point-of-care (POC) with a miniature LED fiberoptic photometer. Objectives for Phase II are: 1) validation of method specificity for unconjugated and delta bilirubin; 2) optimization and technical evaluation of the methods; 3) enhancement of the LED photometer; 4) development of a dry reagent application technique for the POC reagents. The proposed enzymatic assay technology intends to replace the current diazo field methods, which originate in the 19th century, are operationally complex, unstandardized, and have a dismal record of performance. PROPOSED COMMERCIAL APPLICATIONS: In the US alone, close to 250 million tests for total or direct bilirubin are ordered each year. Total billings for bilirubin testing are in excess of $2 billion. The annual economic loss due to liver disease, including alcohol and drug related components, has been estimated at $40 billion. Each year, 40,000 people die from chronic liver disease, and about 300,000 are hospitalized. Assays for total and direct bilirubin are need in 6,000 neonatal care units for the 4 million babies born each year. Current laboratory methods give extremely variable results, and a reliable point- of-care methods give extremely variable results, and reliable point-of-care method is unavailable. The proposed enzymatic methods may help to combat these deficiencies.

该公司已提议开发针对总胆红素、“直接”胆红素和结合胆红素的新型酶特异性测定方法。来自疣状漆斑菌的胆红素氧化酶（胆红素，氧化还原酶，EC.1.3.3.5）将胆红素氧化为胆绿素和更高的氧化产物。通过测量由于内源性胆红素消失而导致的 OD460 减少来监测反应。试剂中存在可回收的铁氰化物可加速信号进程并使其线性化，并增强试剂稳定性。这减少了酶的需求量并促进对分析设备的适应性。在第一阶段，我们已经证明了试剂对其目标胆红素组分具有良好的特异性。开发了原型方法用于：1）在集中式临床分析仪上分析稀释的血清样本； 2) 使用微型 LED 光纤光度计在现场护理 (POC) 测定未稀释的微型样本。第二阶段的目标是： 1) 验证非结合胆红素和 δ 胆红素的方法特异性； 2）方法的优化和技术评估； 3）LED光度计的增强； 4）开发POC试剂的干试剂应用技术。所提出的酶测定技术旨在取代当前的重氮场方法，该方法起源于 19 世纪，操作复杂、不标准化，并且性能记录不佳。拟议的商业应用：仅在美国，每年就订购近 2.5 亿份总胆红素或直接胆红素测试。胆红素检测的总费用超过 20 亿美元。肝病（包括酒精和药物相关成分）造成的年度经济损失估计达 400 亿美元。每年有 40,000 人死于慢性肝病，约 300,000 人住院治疗。 6,000 个新生儿护理单位需要对每年出生的 400 万婴儿进行总胆红素和直接胆红素检测。当前的实验室方法给出了极其可变的结果，并且可靠的护理点方法给出了极其可变的结果，并且可靠的护理点方法不可用。所提出的酶方法可能有助于克服这些缺陷。