CN101169372A - Glutamate dehydrogenase diagnosis reagent kit and glutamate dehydrogenase activity and concentration determination method - Google Patents
Glutamate dehydrogenase diagnosis reagent kit and glutamate dehydrogenase activity and concentration determination method Download PDFInfo
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- CN101169372A CN101169372A CNA2006100968906A CN200610096890A CN101169372A CN 101169372 A CN101169372 A CN 101169372A CN A2006100968906 A CNA2006100968906 A CN A2006100968906A CN 200610096890 A CN200610096890 A CN 200610096890A CN 101169372 A CN101169372 A CN 101169372A
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Abstract
The invention relates to a glutamic dehydrogenase diagnostic reagent box utilizing enzyme-colorimetric techniques, meanwhile, the invention also relates to a method for detecting the activity consistence of the glutamic dehydrogenase, and the compositions and the component of reagent, and belongs to the technical field of medical examination and determination. The reagent box provided by the invention contains buffer solution, reduced coenzyme, 2-ketoglutarate, sal ammoniac and stabilizer. The sample is mixed with the reagent at certain volume ratio to impel the series of enzymatic reaction between the sample and the reagent, and the reactant is then put under an analyzer of visible light/ ultraviolet ray to detect the descending speed of absorbency at the 340nm of the main wavelength, thus, the activity consistence of the glutamic dehydrogenase can be calculated. The invention can completely obtain the needed test result with the help of the analyzer of visible light/ultraviolet ray.
Description
Technical field
The present invention relates to a kind of glutamte dehydrogenase diagnostic kit, the invention still further relates to the method for measuring the glutamte dehydrogenase active concentration simultaneously, belong to medical test determination techniques field.
Background technology
Glutamte dehydrogenase is a kind of enzyme that mainly is present in the cell mitochondrial matrix, and is the highest with liver content, is organs such as kidney, pancreas, brain, mucous membrane of small intestine and heart secondly.It is reported that the glutamte dehydrogenase concentration in the liver is 17 times in the cardiac muscle, 80 times of skeletal muscle, 28 times of pancreas.Because the liver specificity of glutamte dehydrogenase, the change of enzymatic activity can reflect the variation of liver function to a certain extent in the peripheral blood, so be considered to a sensitive indicator of hepatic lesion.The mensuration of serum glutaminic acid dehydrogenasa mainly adopts continuous monitoring method at present.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of enzymic colorimetric (EnzymaticColorimetric Method) technology of utilizing, continuous monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for glutamte dehydrogenase active concentration, simultaneously, the present invention also will provide in order to realize the glutamte dehydrogenase diagnostic kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carry out the glutamte dehydrogenase active concentration determination on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Glutamte dehydrogenase method for measuring active concentration principle of the present invention is as follows:
This method is used glutamte dehydrogenase (Glutamate dehydrogenase; EC 1.4.1.2; EC 1.4.1.3) enzymatic reaction continuous monitoring method.The reaction of glutamte dehydrogenase enzymolysis ALPHA-KG, simultaneously reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into coenzyme (not having absorption peak at the 340nm place), thereby measured the speed that reduced coenzyme descends in 340nm place absorbance, by measuring the speed that 340nm place absorbance descends, can calculate the active concentration size of glutamte dehydrogenase.
Experiment shows, takes all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, no matter is single agent, two agent or three doses, and the glutamte dehydrogenase diagnostic reagent of the present invention of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Sal-ammoniac 30mmol/L
2-oxoglutaric acid ester 20mmol/L
Glutamte dehydrogenase diagnostic kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, reduced coenzyme, 2-oxoglutaric acid ester, sal-ammoniac.
Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Also above-mentioned single agent reagent can be made into following pair of agent reagent:
Reagent 1
Damping fluid, stabilizing agent, reduced coenzyme.
Reagent 2
Damping fluid, 2-oxoglutaric acid ester, sal-ammoniac.
Reduced coenzyme, 2-oxoglutaric acid ester, the position of sal-ammoniac in reagent 1 or reagent 2 can not limit.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Above-mentioned single agent reagent can also be made into following three doses of reagent:
Reagent 1
Damping fluid, stabilizing agent, reduced coenzyme.
Reagent 2
Damping fluid, 2-oxoglutaric acid ester.
Reagent 3
Damping fluid, sal-ammoniac.
Reduced coenzyme, 2-oxoglutaric acid ester, the position of sal-ammoniac in reagent 1, reagent 2 or reagent 3 can not limit.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
No matter be single agent, two agent or three doses, the present invention measures the method for glutamte dehydrogenase active concentration, and its reduced coenzyme can be a kind of among NADPH, NADH or the thio-NADH.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one
The glutamte dehydrogenase diagnostic reagent of present embodiment is a single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Sal-ammoniac 30mmol/L
2-oxoglutaric acid ester 20mmol/L
Reagent divides the bottle of packing into after all dissolving and preparing, and carries out freeze drying, makes powdered reagent; Before the use, add pure water, use after redissolving.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested glutamte dehydrogenase sample and reagent is 1/25, the Direction of Reaction is negative reaction (reaction descends), about about 1 minute of time delay, about 2 minutes detection times, theoretical k value-4180.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 340nm absorbance descends, thereby calculates the active concentration size of glutamte dehydrogenase.
Embodiment two
The glutamte dehydrogenase diagnostic reagent of present embodiment is a double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Sal-ammoniac 20mmol/L
2-oxoglutaric acid ester 30mmol/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid double reagent, can directly use.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested glutamte dehydrogenase sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is negative reaction (reaction descends), about about 1 minute of time delay, about 2 minutes detection times, theoretical k value-2170.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 340nm absorbance descends, thereby calculates the active concentration size of glutamte dehydrogenase.
Embodiment three
The glutamte dehydrogenase diagnostic reagent of present embodiment is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Sal-ammoniac 50mmol/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
2-oxoglutaric acid ester 10mmol/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring the glutamte dehydrogenase active concentration, on automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested glutamte dehydrogenase sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, and the Direction of Reaction is negative reaction (reaction descends), about about 1 minute of time delay, about 2 minutes detection times, theoretical k value-2170.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 340nm absorbance descends, thereby calculates the active concentration size of glutamte dehydrogenase.
The applicant adopts other various reduced form chromogens combinations of putting down in writing in the above summary of the invention all can reach purpose of the present invention through experimental verification, in view of situation such as determination step and above embodiment roughly the same, do not separately enumerate.
In a word, experiment showed, and adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully, and highly sensitive, degree of accuracy good, and is easy to utilize.
Claims (6)
1. glutamte dehydrogenase method for measuring active concentration that utilizes the enzymic colorimetric technology, its method principle is as follows:
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the speed that predominant wavelength 340nm absorbance descends, calculate the active concentration size result of glutamte dehydrogenase.
2. glutamte dehydrogenase diagnostic kit, principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---50mmol/L
Reduced coenzyme 0.1-0.35mmol/L
Sal-ammoniac 1---50mmol/L
2-oxoglutaric acid ester 1---50mmol/L
It is characterized in that: kit can be a dry powder, and use the back that is dissolved in water before use;
Also can be mixed with liquid reagent, directly use.
3. according to the described glutamte dehydrogenase diagnostic kit of claim 2, it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, 2-oxoglutaric acid ester, sal-ammoniac.
4. according to the described glutamte dehydrogenase diagnostic kit of claim 2, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, 2-oxoglutaric acid ester, sal-ammoniac; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme; Reagent 2 is made up of damping fluid, 2-oxoglutaric acid ester, sal-ammoniac.Reduced coenzyme, 2-oxoglutaric acid ester, the position of sal-ammoniac in reagent 1 or reagent 2 can not limit.
5. according to the described glutamte dehydrogenase diagnostic kit of claim 2, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, 2-oxoglutaric acid ester, sal-ammoniac; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme; Reagent 2 is made up of damping fluid, 2-oxoglutaric acid ester; Reagent 3 is made up of damping fluid, sal-ammoniac.Reduced coenzyme, 2-oxoglutaric acid ester, the position of sal-ammoniac in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described glutamte dehydrogenase diagnostic kit of claim 2, it is characterized in that: also comprise stabilizing agent 1-4000mmol/L or 0.1%-100% volume ratio.Described stabilizing agent is: ammonium sulfate (Ammonia Sulfate), glycerine (Glycerol), propylene glycol (PropyleneGlycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100968906A CN101169372A (en) | 2006-10-24 | 2006-10-24 | Glutamate dehydrogenase diagnosis reagent kit and glutamate dehydrogenase activity and concentration determination method |
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| CNA2006100968906A CN101169372A (en) | 2006-10-24 | 2006-10-24 | Glutamate dehydrogenase diagnosis reagent kit and glutamate dehydrogenase activity and concentration determination method |
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| CN101169372A true CN101169372A (en) | 2008-04-30 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104422666A (en) * | 2013-09-09 | 2015-03-18 | 中国科学院沈阳应用生态研究所 | Analysis method for detecting activity of glutamate dehydrogenase in soil |
| CN107782683A (en) * | 2016-08-27 | 2018-03-09 | 山东博科生物产业有限公司 | A kind of glutamate dehydrogenase enzyme detection kit |
-
2006
- 2006-10-24 CN CNA2006100968906A patent/CN101169372A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104422666A (en) * | 2013-09-09 | 2015-03-18 | 中国科学院沈阳应用生态研究所 | Analysis method for detecting activity of glutamate dehydrogenase in soil |
| CN107782683A (en) * | 2016-08-27 | 2018-03-09 | 山东博科生物产业有限公司 | A kind of glutamate dehydrogenase enzyme detection kit |
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Open date: 20080430 |