Use of inhibitors of color generation in chromogenic assays
First Claim
1. In a diagnostic reagent system for the detection of an analyte selected from the group consisting of glucose, cholesterol, triglyceride and uric acid consisting essentially of an oxidase selective for the analyte to be detected, a buffer, a chromogenic oxidation indicator which generates a colored response upon being oxidized together with peroxidase or a perioxidatively active substance, the improvement which comprises an inhibitor of color generation which is selected from the group consisting of 1,4-disubstituted semicarbazides, 1,4-disubstituted thiosemicarbazides and 1,5-disubstituted carbazides and thiosemicarbizides characterized by the formula:
- ##STR7## where X is oxygen or sulfur, R is alkyl or 1-6 carbon atoms, allyl, benzyl, aryl or NH-aryl2 where aryl2 is the same as or different than aryl, which inhibitor reduces the intensity of the color generated by the oxidation of the chromogenic indicator thereby facilitating the visual and instrumental color discrimination between differing levels of analyte in the aqueous system.
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Abstract
Disclosed is a novel diagnostic reagent system for the detection of hydroperoxides or substances which react with peroxidatively active substances resulting in the liberation of hydroperoxides which reagent system comprises a chromogenic oxidation indicator and a peroxidase or a peroxidatively active substance, together with an inhibitor of color generation selected from the group of 1,4-disubstituted semicarbazides, thiosemicarbazides and 1,5-disubstituted carbazides as inhibitors of color generation. By inhibiting the generation of color formed by oxidation of the chromogenic indicator, the indicator'"'"'s usefulness in discriminating between varying concentrations of analyte is enhanced.
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Citations
24 Claims
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1. In a diagnostic reagent system for the detection of an analyte selected from the group consisting of glucose, cholesterol, triglyceride and uric acid consisting essentially of an oxidase selective for the analyte to be detected, a buffer, a chromogenic oxidation indicator which generates a colored response upon being oxidized together with peroxidase or a perioxidatively active substance, the improvement which comprises an inhibitor of color generation which is selected from the group consisting of 1,4-disubstituted semicarbazides, 1,4-disubstituted thiosemicarbazides and 1,5-disubstituted carbazides and thiosemicarbizides characterized by the formula:
- ##STR7## where X is oxygen or sulfur, R is alkyl or 1-6 carbon atoms, allyl, benzyl, aryl or NH-aryl2 where aryl2 is the same as or different than aryl, which inhibitor reduces the intensity of the color generated by the oxidation of the chromogenic indicator thereby facilitating the visual and instrumental color discrimination between differing levels of analyte in the aqueous system.
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 17)
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11. In a diagnostic reagent system for the detection of an analyte in an aqueous system which analyte is selected from the group consisting of glucose, cholesterol, triglyceride and uric acid which reagent system contains an oxidase specific for the analyte whose presence or concentration is being sought, a chromogenic oxidation indicator which generates a colored response upon being oxidized, a buffer and a peroxidase the improvement which consists essentially of the inclusion in such reagent system of an inhibitor of color generation which is a 1,4-disubstituted semicarbazide of the formula:
- ##STR9## wherein R1 is an electron withdrawing group and R2 is allyl, benzyl, or alkyl of 1-6 carbon atoms.
- View Dependent Claims (12, 13, 14, 15)
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16. A diagnostic reagent system for the detection of glucose in an aqueous medium which reagent system comprises glucose oxidase, a buffer, a peroxidase and a substituted benzidine as chromogenic indicator together with a di-substituted semicarbazide of the formula:
- ##STR10## wherein r2 is alkyl of 1-6 carbon atoms, allyl or benzyl and R1 is an electron withdrawing group, said semicarbazide being present in sufficient amount to inhibit the color formed by oxidation of the substituted benzidiene to facilitate discrimination of the color intensity between different degrees of oxidation of the benzidine.
- 18. A reagent pad for the detection of glucose in urine which comprises an absorbant support having absorbed therein glucose oxidase, a buffer, a peroxidase, a benzidine chromogenic indicator and 4-allyl-1-(2-substituted phenyl)semicarbazide.
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20. 1,4-disubstituted semicarbazide compounds of the formula:
- ##STR11## wherein R is alkyl of 1-6 carbon atoms, allyl or benzyl and Ar is an electron withdrawing heterocyclic compound or phenyl substituted with an electron withdrawing group.
- View Dependent Claims (21, 22, 23)
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24. Semicarbazides of claim 209 selected from the group consisting of 4-methyl-1-phenylsemicarbazide, 4-allyl-1-(4-fluorophenyl)semicarbazide, 4-allyl-1-(4-chlorophenyl)-semicarbazide, 4-benzyl-1-phenylsemicarbazide, 4-benzyl-1-(2-pyridyl)semicarbazide, 4-allyl-1-(2-fluorophenyl)-semicarbazide, 4-allyl-1-(2-chlorophenyl)semicarbazide, 4-allyl-1-(4-methoxyphenyl)semicarbazide, 4-allyl-1-(4-bromophenyl)-semicarbazide, 4-allyl-1-(2-bromo-phenyl)semicarbazide, 4-allyl-1-(4-tolyl)semicarbazide, 4-allyl-1-(2-tolyl)semicarbazide, 4-allyl-1-(3-bromophenyl)semicarbazide and 4-allyl-1-(3-nitrophenyl)semicarbazide.
Specification