Abstract A novel substrate solution, dye-coupling composition and method for determining gamma-glutamyl transpeptidase in biological fluids have been developed. The substrate solution is an aqueous buffered solution containing an L-gamma-glutamyl-p-nitroanilide substrate, glycylglycine, sodium nitrite, and optionally, magnesium chloride hexahydrate. The dye-coupling composition for use in conjunction with the substrate solution comprises a mineral acid reagent solution of 8-hydroxy quinoline sulfate containing a water soluble, nonionic emulsifier, adjusted to a pH of between 0.8 and 1.3. In the determination of gamma-glutamyl transpeptidase, the biological test fluid is incubated with the substrate solution for a predetermined period of time; when incubation is completed, the dye-coupling composition is added to initiate the dye-forming reaction; an alkaline solution which will impart a pH of between 10.5 and 12 is then added to promote color development. The final alkaline solution optionally may contain ethylenediaminetetraacetic acid, tetrasodium salt. The optical density of the total reaction solution is a measure of gamma-glutamyl transpeptidase concentration. The method of the invention is accurate and approximately 4.5 times more sensitive than the standard test method currently being used.
A DIAGNOSTIC COMPOSITION FOR THE DETERMINATION OF GAMMA-GLUTAMYL TRANSPEPTIDASE CONCENTRATION IN BIOLOGICAL FLUIDS COMPRISING AN AQUEOUS SUBSTRATE SOLUTION CONTAINING: A. AT LEAST ABOUT 45 MICROMOLES OF L-GAMMA-GLUTAMYL-PNITROANILIDE PER MILLILITER OF BIOLOGICAL FLUID, B. AT LEAST 400 MICROMOLED OF GLYCGLYCINE PER MILLILITER OF BIOLOGICAL FLUID, AND C. AT LEAST 36.2 MICROMOLED OF SODIUM NITRITE PER MILLILITER OF BIOLOGICAL FLUID, SAID AQUEOUS SOLUTION BEING BUFFERED TO MAINTAIN A PH OF FROM ABOUT 7.9 TO ABOUT 8.4.
Claim 2
Dependent Claim
74 words
A DIAGNOSTIC COMPOSITION FOR THE DETERMINATION OF GAMMA-GLUTAMYL TRANSPEPTIDASE CONCENTRATION IN BIOLOGICAL FLUIDS COMPRISING AN AQUEOUS SUBSTRATE SOLUTION CONTAINING: A. AT LEAST ABOUT 45 MICROMOLES OF L-GAMMA-GLUTAMYL-PNITROANILIDE PER MILLILITER OF BIOLOGICAL FLUID, B. AT LEAST 400 MICROMOLED OF GLYCGLYCINE PER MILLILITER OF BIOLOGICAL FLUID, AND C. AT LEAST 36.2 MICROMOLED OF SODIUM NITRITE PER MILLILITER OF BIOLOGICAL FLUID, SAID AQUEOUS SOLUTION BEING BUFFERED TO MAINTAIN A PH OF FROM ABOUT 7.9 TO ABOUT 8.4.
Claim 3
Dependent Claim
24 words
A diagnostic composition according to claim 1 wherein the substrate solution is buffered to a pH of about 8.2 with a (2-amino-2-methyl-propane-1,3-diol)-HC1 buffer system.
Claim 4
Dependent Claim
26 words
A diagnostic composition according to claim 1 wherein the substrate solution is buffered to a pH of about 8.2 with a tris-(hydroxymethyl)amino methane hydrochloride buffer system.
Claim 5
Dependent Claim
26 words
A diagnostic composition according to claim 1 wherein the substrate solution additionally contains at least 100 micromoles of magnesium chloride hexahydrate per milliliter of biological fluid.
Claim 6
Dependent Claim
26 words
A diagnostic composition according to claim 4 wherein the substrate solution is buffered to a pH of about 8.2 with a tris-(hydroxymethyl)amino methane hydrochloride buffer system.