Novel Enzymic Assays for Total and Conjugated Bilirubin

Period of Performance: 09/01/2000 - 08/31/2002

$341K

Phase 2 SBIR

Recipient Firm

Micronix, Inc.
Carmel, IN 46032
Principal Investigator

Abstract

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.