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US 5,846,754 AGranted
Enzyme determination with protease inactivation
AssigneeBayer Aktiengesellschaft
InventorsMichael J. Pugia, Robert J. Schaeper
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AssigneeBayer Aktiengesellschaft
InventorsMichael J. Pugia, Robert J. Schaeper
Estimating the term…
The present invention involves a composition and method for the determination of enzymatic activity or enzyme substrate concentration in a fluid test sample in which the enzyme catalyzes a reaction in a reagent system to provide a detectable response indicative of the concentration of the enzyme or enzyme substrate in the test sample. A measured amount of a protease capable of eliminating the enzymatic activity is included in the reagent composition, so that when a measured amount of test sample is treated, the reaction producing the detectable response will have a definite end point. The invention is particularly useful in conjunction with the determination of leucocytes in urine wherein the leukocytes are lysed to release their human leucocyte elastase which hydrolyzes a chromogenic ester to provide a phenol which then reacts with a diazonium salt to form a colored azo dye.
In a method for the determination of enzymatic activity in a fluid test sample wherein an enzyme catalyzes a reaction in a reagent system to provide a detectable response indicative of the concentration of the enzyme or enzyme substrate in the fluid test sample, the improvement which comprises adding a measured amount of the fluid test sample to a measured amount of a protease capable of inactivity the enzyme responsible for producing the detectable response which does not inactivate the reagent system which provides the detectable response so that the reaction which provides the detectable response ceases after a predetermined time which time is determined by the concentration of the protease in the reagent system.
In a composition for the determination of leukocytes in a measured amount of an aqueous test fluid by measuring the amount of esterase released upon lysis of the leukocytes which composition comprises a diazonium salt and an ester which is hydrolyzed by the esterase to yield a phenol or a pseudophenol which couples with the diazonium salt by a diazonium coupling reaction to form a colored azo dye wherein the intensity of the color is indicative of the concentration of leukocytes in the aqueous test fluid, the improvement which comprises including a measured amount of a protease, which does not cleave the ester but cleaves the esterase in the composition, so that the color forming reaction between the diazonium salt and phenol or pseudophenol ceases after a predetermined time which time is determined by the concentration of the protease in the composition.
A test strip for the determination of enzymatic activity or amount of enzyme substrate in a fluid test sample which comprises an absorbent carrier as the strip material which absorbs a measured amount of the fluid test sample when contacted therewith having absorbed therein a buffering agent and an enzyme substrate which will provide a colored detectable response upon exposure to the enzyme in the fluid test sample together with a measured amount of a protease which inactivates the enzymatic activity but does not cause the detectable response in the enzyme substrate, so that the reaction which provides the colored detectable response ceases after a predetermined time which time is determined by the concentration of the protease in the reagent system.
Dependencies are inferred from the claim text.