CN101324572A - Glutamic acid diagnosis/determination reagent kit and method for determining glutamic acid concentration - Google Patents
Glutamic acid diagnosis/determination reagent kit and method for determining glutamic acid concentration Download PDFInfo
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- CN101324572A CN101324572A CNA2007100233961A CN200710023396A CN101324572A CN 101324572 A CN101324572 A CN 101324572A CN A2007100233961 A CNA2007100233961 A CN A2007100233961A CN 200710023396 A CN200710023396 A CN 200710023396A CN 101324572 A CN101324572 A CN 101324572A
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- glyceraldehyde
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- phosphate
- glutamic acid
- ammonia
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Abstract
The invention relates to a kit for diagnosing/mensurating glutamic acid by utilizing the technologies of the enzymic colorimetric method and the enzyme linked immunosorbent assay (ELISA). The invention further relates to a method, a principle and the composition and the components of a reagent for mensurating the concentration of the glutamic acid, and belongs to the technology field of medical/food inspection and measurement. The main components of the kit include a buffer solution, coenzyme, ammonia, adenyl pyrophosphate, glyceraldehyde-3-phosphate, glutamine synthetase, glyceraldehyde-3-phosphate dehydrogenase and a stabilizer. Through mixing a sample and the reagent by a certain volume ratio, an enzymatic reaction occurs, then the reactant is placed under an ultraviolet/visible light analyzer, and the degree/velocity of the increase in absorbance at 340nm of the dominant wavelength is detected, thereby mensurating the concentration of the glutamic acid.
Description
Technical field
The present invention relates to a kind of glutamic acid diagnosis/mensuration kit, the invention still further relates to the method for measuring aminoglutaric acid concentration simultaneously, belong to medical science/Food Inspection determination techniques field.
Background technology
State Standard of the People's Republic of China GB5009-2003 monosodium glutamate hygienic standard analytical approach has: 1. polarimeter method 2. formol titration 3. Kjeldahls.Monosodium glutamate means that with grain and goods be the sodium glutamate that raw material is purified through fermentation.
Enzymatic analysis is the analytical approach of the glutamate of generally acknowledging in the world, organized by a plurality of internal authorities or mechanism adopts: as IFU (international fruit juice association of producers), AIJN (European Union's Juice and beverage production person alliance), and MEBAK, OICC, VDLUFA etc. organize widely and praise highly, and are defined as standard detecting method.Be written into Australia, Holland, Germany, the food law of a plurality of countries such as Switzerland.Domestic also have the man fruit juice of number, drinks processing enterprise to use the enzyme process kit as examination criteria.
Have osteoclast that decomposes sclerotin and the Gegenbaur's cell that forms bone in the biosome, the effect of the following two kinds of cells of normal condition is keeping good balance.If balance is broken, osteoclast is too active, will cause osteoporosis.Contain glutamic acid in the osteoclast, and the albumen of a kind of " glutamate transporter " by name can be discharged glutamic acid osteoclast, the glutamic acid meeting that is " released " and the receptors bind of cell surface, the enzyme in the active cell suppresses the effect that osteoclast decomposes sclerotin.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of enzymic colorimetric (EnzymaticColorimetric Method) and enzyme (even) united method (Couple Reaction) technology utilized, metering/continuous monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for aminoglutaric acid concentration, simultaneously, the present invention also will provide in order to realize the glutamic acid diagnosis/mensuration kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carrying out aminoglutaric acid concentration on the automatic clinical chemistry analyzer measures, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Aminoglutaric acid concentration assay method principle of the present invention is as follows:
Glutamic acid+ammonia+adenosine triphosphate
Glutamine synthelaseGlutamine+
Adenosine diphosphate+phosphate radical
Phosphate radical+glyceraldehyde-3-phosphate+coenzyme
Glyceraldehyde-3-phosphate dehydrogenase
Glyceric acid-1,3-bis phosphoric acid+reduced coenzyme
This method is used glutamine synthelase (glutamine synthetase; EC 6.3.1.2) coupling glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase; EC1.2.1.59) enzyme ' s reaction speeding colourimetry/end-point method.The reaction of glutamine synthelase enzymolysis glutamic acid produces phosphate radical, effect by the coupling glyceraldehyde-3-phosphate dehydrogenase again, coenzyme (not having absorption peak at the 340nm place) reduces the most at last becomes reduced coenzyme (absorption peak being arranged at the 340nm place), thereby measured degree/speed that reduced coenzyme rises in 340nm place absorbance, by measuring degree/speed that 340nm place absorbance rises, can calculate the concentration of glutamic acid.
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 glutamic acid diagnosis/mensuration kit of the present invention of following composition relation is ideal comparatively:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Ammonia 30mmol/L
Adenosine triphosphate 10mmol/L
Glyceraldehyde-3-phosphate 10mmol/L
Glutamine synthelase 10000U/L
Glyceraldehyde-3-phosphate dehydrogenase 16000U/L
Glutamic acid diagnosis/mensuration kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, ammonia, adenosine triphosphate, glyceraldehyde-3-phosphate, glutamine synthelase, glyceraldehyde-3-phosphate dehydrogenase.
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, coenzyme, ammonia, adenosine triphosphate, glyceraldehyde-3-phosphate.
Reagent 2
Damping fluid, stabilizing agent, glutamine synthelase, glyceraldehyde-3-phosphate dehydrogenase.
Coenzyme, ammonia, adenosine triphosphate, glyceraldehyde-3-phosphate, glutamine synthelase, the position of glyceraldehyde-3-phosphate dehydrogenase 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, coenzyme, ammonia, adenosine triphosphate, glyceraldehyde-3-phosphate.
Reagent 2
Damping fluid, stabilizing agent, glyceraldehyde-3-phosphate dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, glutamine synthelase.
Coenzyme, ammonia, adenosine triphosphate, glyceraldehyde-3-phosphate, glutamine synthelase, the position of glyceraldehyde-3-phosphate dehydrogenase 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 aminoglutaric acid concentration, and its coenzyme can be NADP
+, NAD
+Or thio-NAD
+In a kind of.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one
Glutamic acid diagnosis/mensuration the reagent of present embodiment is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Ammonia 30mmol/L
Adenosine triphosphate 10mmol/L
Glyceraldehyde-3-phosphate 10mmol/L
Glutamine synthelase 10000U/L
Glyceraldehyde-3-phosphate dehydrogenase 16000U/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≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested glutamic acid sample and reagent is 1/25, the Direction of Reaction is positive reaction (reaction of rising), about about 0 minute of time delay is about 5 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects degree/speed that predominant wavelength 340nm absorbance rises, thereby calculates the concentration of glutamic acid.
Embodiment two
Glutamic acid diagnosis/mensuration the reagent of present embodiment is double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Ammonia 30mmol/L
Adenosine triphosphate 10mmol/L
Glyceraldehyde-3-phosphate 10mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glutamine synthelase 10000U/L
Glyceraldehyde-3-phosphate dehydrogenase 16000U/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≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested glutamic acid sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is positive reaction (reaction of rising), about about 0 minute of time delay is about 5 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects degree/speed that predominant wavelength 340nm absorbance rises, thereby calculates the concentration of glutamic acid.
Embodiment three
Glutamic acid diagnosis/mensuration the reagent of present embodiment is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Ammonia 30mmol/L
Adenosine triphosphate 10mmol/L
Glyceraldehyde-3-phosphate 10mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glyceraldehyde-3-phosphate dehydrogenase 16000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glutamine synthelase 10000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring aminoglutaric acid concentration, on automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested glutamic acid sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, the Direction of Reaction is positive reaction (reaction of rising), and about about 0 minute of time delay is about 5 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects degree/speed that predominant wavelength 340nm absorbance rises, thereby calculates the concentration of glutamic acid.
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. the method for measurement of concentration of the glutamic acid of enzymic colorimetric and enzyme-linked method, its method principle is as follows:
Glutamic acid+ammonia+adenosine triphosphate
Glutamine synthelaseGlutamine+
Adenosine diphosphate+phosphate radical
Phosphate radical+glyceraldehyde-3-phosphate+coenzyme
Glyceraldehyde-3-phosphate dehydrogenase
Glyceric acid-1,3-bis phosphoric acid+reduced coenzyme
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect degree/speed that predominant wavelength 340nm absorbance rises, calculate the concentration measurement result of glutamic acid.
2. glutamic acid diagnosis/mensuration kit, principal ingredient comprises:
Damping fluid 20-500mmol/L
Stabilizing agent 1-4000mmol/L
DPN diphosphopyridine nucleotide-6mmol/L
Ammonia 1-50mmol/L
Adenosine triphosphate 1-50mmol/L
Glyceraldehyde-3-phosphate 1-50mmol/L
Glutamine synthelase 1000-80000U/L
Glyceraldehyde-3-phosphate dehydrogenase 1000-80000U/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.Ammonia can use any ammonia salt.
3. according to the described glutamic acid diagnosis/mensuration of claim 2 kit, it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, coenzyme, ammonia, adenosine triphosphate, glyceraldehyde-3-phosphate, glutamine synthelase, glyceraldehyde-3-phosphate dehydrogenase.
4. according to the described glutamic acid diagnosis/mensuration of claim 2 kit, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, coenzyme, ammonia, adenosine triphosphate, glyceraldehyde-3-phosphate, glutamine synthelase, glyceraldehyde-3-phosphate dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, ammonia, adenosine triphosphate, glyceraldehyde-3-phosphate; Reagent 2 is made up of damping fluid, stabilizing agent, glutamine synthelase, glyceraldehyde-3-phosphate dehydrogenase.Coenzyme, ammonia, adenosine triphosphate, glyceraldehyde-3-phosphate, glutamine synthelase, the position of glyceraldehyde-3-phosphate dehydrogenase in reagent 1 or reagent 2 can not limit.
5. according to the described glutamic acid diagnosis/mensuration of claim 2 kit, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, coenzyme, ammonia, adenosine triphosphate, glyceraldehyde-3-phosphate, glutamine synthelase, glyceraldehyde-3-phosphate dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, ammonia, adenosine triphosphate, glyceraldehyde-3-phosphate; Reagent 2 is made up of damping fluid, stabilizing agent, ammonia, glyceraldehyde-3-phosphate dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, glutamine synthelase.Coenzyme, ammonia, adenosine triphosphate, glyceraldehyde-3-phosphate, glutamine synthelase, the position of glyceraldehyde-3-phosphate dehydrogenase in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described glutamic acid diagnosis/mensuration of claim 2 kit, 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 (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
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|---|---|---|---|
| CNA2007100233961A CN101324572A (en) | 2007-06-13 | 2007-06-13 | Glutamic acid diagnosis/determination reagent kit and method for determining glutamic acid concentration |
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|---|---|---|---|
| CNA2007100233961A CN101324572A (en) | 2007-06-13 | 2007-06-13 | Glutamic acid diagnosis/determination reagent kit and method for determining glutamic acid concentration |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102298032A (en) * | 2010-06-25 | 2011-12-28 | 苏州艾杰生物科技有限公司 | Method for determining glycine and glycine determination kit |
-
2007
- 2007-06-13 CN CNA2007100233961A patent/CN101324572A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102298032A (en) * | 2010-06-25 | 2011-12-28 | 苏州艾杰生物科技有限公司 | Method for determining glycine and glycine determination kit |
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Open date: 20081217 |