NOVEL ENZYMIC ASSAYS FOR TOTAL AND CONJUGATED BILIRUBIN

该公司提出了开发新的酶特异性总胆红素、直接胆红素和结合胆红素的检测方法。来自寻常疣霉菌的胆红素氧化酶(胆红素，氧化还原酶，EC.1.3.3.5)将胆红素氧化成胆红素和更高级的氧化产物。通过测量由于内源性胆红素消失而导致的OD460的减少来监测反应。试剂中可回收铁氰化物的存在加速和线性化了信号进程，并提高了试剂的稳定性。这减少了酶的需求量并促进了对分析设备的适应性。在第一阶段中，我们已经证明了试剂对其靶向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样本；2)在护理点(POC)用微型LED光纤光度计测定未稀释的微量样本。第二阶段的目标是：1)验证未结合胆红素和增量胆红素的方法特异性；2)方法的优化和技术评估；3)增强LED光度计；4)开发POC试剂的干试剂应用技术。拟议的酶分析技术旨在取代目前的重氮法，这些方法起源于19世纪，操作复杂，不标准化，性能记录令人沮丧。拟议的商业应用：仅在美国，每年就订购了近2.5亿次总胆红素或直接胆红素测试。胆红素检测的总账单超过20亿美元。每年因肝病造成的经济损失估计为400亿美元，其中包括与酒精和药物相关的成分。每年有4万人死于慢性肝病，约30万人住院治疗。每年出生的400万婴儿需要在6000个新生儿护理病房进行总胆红素和直接胆红素的检测。目前的实验室方法给出的结果千差万别，可靠的护理点法给出的结果千差万别，而可靠的护理点法是不可用的。建议的酶促方法可能有助于克服这些不足。

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.