Creatine kinase isoenzyme electrophoretic technique and improved creatine kinase isoenzyme reagent for use therein
AssigneeBeckman Instruments, Inc.
InventorRobert T. Y. Lee
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Abstract
A creatine kinase reagent of the type comprising a buffer, creatine phosphate, adenosine diphosphate, D-glucose, hexokinase, glucose-6-phosphate dehydrogenase, a thiol compound, magnesium ion, adenosine monophosphate, and a coenzyme selected from a group consisting of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and mixtures thereof. The reagent is characterized in that it further comprises an effective amount of an enzyme selected from a group consisting of phosphogluconate dehydrogenase and phosphogluconate dehydrogenase (decarboxylating). An electrophoretic technique for assaying the relative distribution of creatine kinase characterized in that the above reagent is employed therein.
Claims15
A creatinine kinase reagent of the type comprising a buffer, creatinine phosphate, adenosine diphosphate, D-glucose, hexokinase, glucose-6-phosphate dehydrogenase, a thiol compound, magnesium ion, adenosine monophosphate, and a coenzyme selected from the group consisting of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and mixtures thereof, characterized in that said reagent further comprises an enzyme selected from a group consisting of phosphogluconate dehydrogenase and phosphogluconate dehydrogenase (decarboxylating) in an amount sufficient to react 6-phosphogluconate with said coenzyme in the presence of said enzyme.
An improved electrophoretic technique for assaying the relative distribution of creatine kinase of the type wherein a sample to be assayed is applied to an electrophoretic gel, said electrophoretic gel is electrophoresed, said electrophoresed, electrophoretic gel is contacted with a reagent thereby producing a series of simultaneous reactions between the creatine kinase and the constituents of said reagent, said simultaneous reactions comprising:
- (a) reacting creatine phosphate with adenosine diphosphate in the presence of creatine kinase to form creatine and adenosine triphosphate;
- (b) reacting adenosine triphosphate with D-glucose in the presence of hexokinase to form adenosine diphosphate and glucose-6-phosphate;
- and
- (c) reacting glucose-6-phosphate with a coenzyme selected from a group consisting of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and mixtures thereof in the presence of glucose-6-phosphate dihydrogenase to form 6-phosphogluconate and the reduced form of said coenzyme;
- and measuring the reduced coenzyme present in the resulting electrophoresed pattern to determine the relative distribution of said creatine kinase in the assayed sample;
- the electrophoretic technique being improved in that said 6-phosphogluconate is reacted with said coenzyme in the presence of an enzyme selected from a group consisting of phosphogluconate dehydrogenase and phosphogluconate dehydrogenase (decarboxylating) to produce an additional mole of said reduced coenzyme, thereby increasing the sensitivity of the creatine kinase electrophoretic technique.
Dependencies are inferred from the claim text.