Bioluminescence methods for enzymatic determinations
AssigneeThe United States of America as represented by the United States Department of Energy
InventorsWilliam D. Bostick, Mark S. Denton, Stanley R. Dinsmore
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Abstract
An enzymatic method for continuous, on-line and rapid detection of diagnostically useful biomarkers, which are symptomatic of disease or trauma-related tissue damage, is disclosed. The method is characterized by operability on authentic samples of complex biological fluids which contain the biomarkers.
Claims6
A nonreferenced method for continuous, on-line and rapid detection of diagnostically useful biomarkers, which are symptomatic of disease or trauma-related tissue damage, said method characterized by operability on an authentic sample of complex biological fluids, containing a plurality of said biomarkers, comprising, in combination, the steps of:
- contacting a predetermined amount of the sample with an enzymatically effective amount of reactants to generate a specific bioluminescent emission correlatable to the concentration of a specific diagnostically useful biomarker isolated by liquid chromatography from said biomarker plurality;
- and
- monitoring the emission intensity to determine the specific biomarker concentration in a fluorometer characterized by the absence of an excitation source and filters.
A nonreferenced and automated method for quantifying at least one of a complex mixture of tissue-specific isoenzymes in an aqueous media comprising the steps of:
- (a) resolving the complex mixture into discernable isoenzyme components by liquid chromatography;
- (b) contacting a predetermined amount of the resolved components with at least one or more reactants in an amount effective to produce an ordered sequence of reactions wherein:
- (A) in a first reaction, at least one resolved component produces as a product the reduced form of NAD or NADP;
- (B) in a second reaction, said reduced form is oxidized in the presence of flavin oxidoreductase to produce the reduced form of riboflavin and NAD or NADP;
- and
- (C) in a third reaction, the reduced form of riboflavin is oxidized in the presence of oxygen, dodecanal, and bacterial luciferase to produce a bioluminescent emission accurately confirming the presence of the at least one resolved component;
- and
- (c) correlating said emission to the concentration of the at least one resolved component.
Dependencies are inferred from the claim text.