NOVEL ENZYMATIC ASSAYS FOR TOTAL AND CONJUGATED BILIRUBI

该公司已提出开发新的酶特异性测定总胆红素、“直接胆红素”和结合胆红素的方法。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的消失导致的OD 460的降低来监测反应。试剂中可回收的铁氰化物的存在加速和线性化信号进展并增强试剂稳定性。这减少了所需的酶的量，并有利于分析设备的适应性。在第一阶段，我们已经证明了试剂对其目标胆红素组分的良好特异性。开发的原型方法用于：1）在集中式临床分析仪上分析稀释的血清标本; 2）在床旁（POC）使用微型LED光纤光度计测定未稀释的微量标本。第二阶段的目标是：1）未结合胆红素和δ胆红素的方法专属性验证; 2）方法的优化和技术评价; 3）LED光度计的增强; 4）POC试剂干试剂应用技术的开发。所提出的酶测定技术旨在取代目前的重氮场方法，重氮场方法起源于世纪，操作复杂，不标准化，并且具有令人沮丧的性能记录。建议的商业应用：仅在美国，每年就有近2.5亿例总胆红素或直接胆红素检测。胆红素检测的总账单超过20亿美元。由于肝病，包括酒精和药物相关成分，每年的经济损失估计为400亿美元。每年有4万人死于慢性肝病，约30万人住院治疗。每年有400万名新生儿出生，需要在6,000个新生儿护理单位进行总胆红素和直接胆红素检测。目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。所提出的酶促方法可能有助于克服这些缺陷。

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.