CN101464272A - Glycine diagnosis/measuring reagent kit and glycine concentration determination method - Google Patents
Glycine diagnosis/measuring reagent kit and glycine concentration determination method Download PDFInfo
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- CN101464272A CN101464272A CNA2007101914255A CN200710191425A CN101464272A CN 101464272 A CN101464272 A CN 101464272A CN A2007101914255 A CNA2007101914255 A CN A2007101914255A CN 200710191425 A CN200710191425 A CN 200710191425A CN 101464272 A CN101464272 A CN 101464272A
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- reagent
- coenzyme
- glycine
- stabilizing agent
- glycocoll
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Abstract
The invention relates to a kit for diagnosing/measuring glycine by utilizing the technologies of the doubling amplification method, the enzymic colorimetry and the enzyme linked immunosorbent assay. The invention further relates to a method, a principle and the composition and the components of a reagent for measuring the concentration of the glycine, and belongs to the technical field of medical/food inspection and measurement. The main components of the kit include a buffer solution, coenzyme, inosinic monophosphate, glycine dehydrogenase, guanosine monophosphate reduced enzyme and a stabilizer. Through mixing a sample and the reagent by a certain volume ratio, a series of enzymatic reactions occur, then the reactant is placed under an ultraviolet/visible light analyzer, and the degree/velocity of the increase in absorbance at 340 nm of the dominant wavelength is detected, thereby measuring the active concentration of the glycine.
Description
Technical field
The present invention relates to a kind of glycocoll diagnosis/mensuration kit, the invention still further relates to the method for measuring glycine concentration simultaneously, belong to medical science/Food Inspection determination techniques field.
Background technology
Glycocoll is a kind of important fine-chemical intermediate, is widely used in industries such as agricultural chemicals, medicine, food, chemical fertilizer, feed.Can be divided into food-grade, pharmaceutical grade, feed grade and four kinds of specification products of technical grade according to the preparation technology of glycocoll and the purity of product.
In 20 several amino acids, glycocoll is the simplest a kind of, and it is a kind of composition of constitutive protein matter, is non-essential amino acid." food additives use hygienic standard " regulation, the scope that glycocoll allows to use is flavoring and soymilk, maximum use amount is 1g/kg.The special supervision and check result that State General Administration for Quality Supervision carries out a few days ago shows, domestic part milk beverage enterprise uses the much lower glycocoll of price to replace milk powder in production and processing for improving protein content in the product in violation of rules and regulations, and indivedual enterprises problem is serious.Introduce according to the insiders, use glycocoll to substitute milk powder, can make cost of products reduce half at least.Human body is too much if take in the amount of glycocoll, and the utilization that not only can not be absorbed by the body, and can break human body amino acid whose absorption equilibrium is influenced other amino acid whose absorption causes nutrient imbalance and unhealthful.
The examination criteria of each place all adopts the determining N of amino acid method to measure glycocoll content at present.Newer method adopts HPLC method (derivatization method behind the performance liquid chromatographic column) mensuration glycocoll wherein.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of double TRAP (DoublingMethod) of utilizing, enzymic colorimetric (Enzymatic Colorimetric Method) and coupling method (CoupleReaction) technology, metering/continuous monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for glycine concentration, simultaneously, the present invention also will provide in order to realize the glycocoll diagnosis/mensuration kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carrying out glycine concentration on the automatic clinical chemistry analyzer measures, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Glycine concentration assay method principle of the present invention is as follows:
Glycocoll+water+coenzyme
Glycine dehydrogenaseGlyoxalic acid+ammonia+
Reduced coenzyme+H
+
Inosine list phosphoric acid+ammonia+coenzyme
Guanosine monophosphate reductaseGuanosine monophosphate+
Reducibility coenzyme
This method is used glycine dehydrogenase (glycine dehydrogenase; EC 1.4.1.10) coupling guanosine monophosphate reductase (GMP reductase; EC 1.7.1.7) enzyme ' s reaction speeding colourimetry/end-point method.Glycine dehydrogenase enzymolysis glycine reactant produces ammonia, and simultaneously with coenzyme (not having absorption peak) reduction becoming reduced coenzyme (absorption peak being arranged) at the 340nm place at the 340nm place, in addition, effect by coupling guanosine monophosphate reductase and ammonia again, for the second time again with coenzyme (not having absorption peak) reduction becoming reduced coenzyme (absorption peak being arranged) at the 340nm place at the 340nm place, thereby be able to two times and measure degree/speed that reduced coenzyme rises in 340nm place absorbance, by measuring degree/speed that 340nm place absorbance rises, can calculate the concentration of glycocoll.
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 glycocoll 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
Glycine dehydrogenase 10000U/L
Guanosine monophosphate reductase 12000U/L
Inosine list phosphoric acid 8mmol/L
Glycocoll diagnosis/mensuration kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, glycine dehydrogenase, guanosine monophosphate reductase, inosine list phosphoric acid.
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, inosine list phosphoric acid.
Reagent 2
Damping fluid, stabilizing agent, glycine dehydrogenase, guanosine monophosphate reductase.
Coenzyme, glycine dehydrogenase, guanosine monophosphate reductase, the position of inosine list phosphoric acid 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, inosine list phosphoric acid.
Reagent 2
Damping fluid, stabilizing agent, guanosine monophosphate reductase.
Reagent 3
Damping fluid, stabilizing agent, glycine dehydrogenase.
Coenzyme, glycine dehydrogenase, guanosine monophosphate reductase, the position of inosine list phosphoric acid 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 glycine 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
Glycocoll 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
Glycine dehydrogenase 10000U/L
Guanosine monophosphate reductase 12000U/L
Inosine list phosphoric acid 8mmol/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 glycocoll 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 glycocoll.
Embodiment two
Glycocoll 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
Inosine list phosphoric acid 8mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Glycine dehydrogenase 10000U/L
Guanosine monophosphate reductase 12000U/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 glycocoll 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 glycocoll.
Embodiment three
Glycocoll 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
Inosine list phosphoric acid 8mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Guanosine monophosphate reductase 12000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glycine dehydrogenase 10000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring glycine 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 glycocoll 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 glycocoll.
The applicant adopts other assay methods 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, experimental results show that: adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully---the blank reagent absorbance changes (Δ A/min)≤0.0010; Absorbance time response curve should be the rising curve until terminal point; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 10mmol/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 7%; The coefficient of variation (CV)≤2% of the precision of reagent test (repeatability); The sensitivity of reagent can reach that 0.1038 ± 0.0450 Δ A/mmol/L---the present invention is highly sensitive, degree of accuracy good, and the linear range broadness is enough to easy to utilize.
Claims (6)
1. the method for measurement of concentration of the glycocoll of a double TRAP, enzymic colorimetric and enzyme-linked method, its method principle is as follows:
Glycocoll+water+coenzyme
Glycine dehydrogenaseGlyoxalic acid+ammonia+
Reduced coenzyme+H
+
Inosine list phosphoric acid+ammonia+coenzyme
Guanosine monophosphate reductaseGuanosine monophosphate+
Reducibility 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 glycocoll.
2. glycocoll diagnosis/mensuration kit, principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
DPN diphosphopyridine nucleotide---6mmol/L
Glycine dehydrogenase 1000---80000U/L
Guanosine monophosphate reductase 1000---80000U/L
Inosine list phosphatase 11---50mmol/L
The concentration of reagent composition not necessarily is only limited to above-mentioned scope; Effect is better in this scope, and outside this scope, reagent still can reagentia.
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 glycocoll diagnosis/mensuration of claim 2 kit, it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, coenzyme, glycine dehydrogenase, guanosine monophosphate reductase, inosine list phosphoric acid.
4. according to the described glycocoll diagnosis/mensuration of claim 2 kit, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, coenzyme, glycine dehydrogenase, guanosine monophosphate reductase, inosine list phosphoric acid; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, inosine list phosphoric acid; Reagent 2 is made up of damping fluid, stabilizing agent, glycine dehydrogenase, guanosine monophosphate reductase.Coenzyme, glycine dehydrogenase, guanosine monophosphate reductase, the position of inosine list phosphoric acid in reagent 1 or reagent 2 can not limit.
5. according to the described glycocoll diagnosis/mensuration of claim 2 kit, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, coenzyme, glycine dehydrogenase, guanosine monophosphate reductase, inosine list phosphoric acid; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, inosine list phosphoric acid; Reagent 2 is made up of damping fluid, stabilizing agent, guanosine monophosphate reductase; Reagent 3 is made up of damping fluid, stabilizing agent, glycine dehydrogenase.Coenzyme, glycine dehydrogenase, guanosine monophosphate reductase, the position of inosine list phosphoric acid in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described glycocoll 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101914255A CN101464272A (en) | 2007-12-19 | 2007-12-19 | Glycine diagnosis/measuring reagent kit and glycine concentration determination method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101914255A CN101464272A (en) | 2007-12-19 | 2007-12-19 | Glycine diagnosis/measuring reagent kit and glycine concentration determination method |
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| Publication Number | Publication Date |
|---|---|
| CN101464272A true CN101464272A (en) | 2009-06-24 |
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|---|---|---|---|
| CNA2007101914255A Pending CN101464272A (en) | 2007-12-19 | 2007-12-19 | Glycine diagnosis/measuring reagent kit and glycine concentration determination method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102087265A (en) * | 2009-12-04 | 2011-06-08 | 苏州艾杰生物科技有限公司 | Determination method of glycine and kit for determining glycine |
| CN102087267A (en) * | 2009-12-04 | 2011-06-08 | 苏州艾杰生物科技有限公司 | Method and kit for measuring glycine |
| CN102539717A (en) * | 2010-12-13 | 2012-07-04 | 苏州艾杰生物科技有限公司 | Ammonia (ammonia ion) measurement method and ammonia (ammonia ion) diagnosis/measurement kit |
| CN106644987A (en) * | 2016-12-31 | 2017-05-10 | 湖南永和阳光生物科技股份有限公司 | Circular enzyme detection method for detecting CG (cholyglycine) |
-
2007
- 2007-12-19 CN CNA2007101914255A patent/CN101464272A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102087265A (en) * | 2009-12-04 | 2011-06-08 | 苏州艾杰生物科技有限公司 | Determination method of glycine and kit for determining glycine |
| CN102087267A (en) * | 2009-12-04 | 2011-06-08 | 苏州艾杰生物科技有限公司 | Method and kit for measuring glycine |
| CN102539717A (en) * | 2010-12-13 | 2012-07-04 | 苏州艾杰生物科技有限公司 | Ammonia (ammonia ion) measurement method and ammonia (ammonia ion) diagnosis/measurement kit |
| CN106644987A (en) * | 2016-12-31 | 2017-05-10 | 湖南永和阳光生物科技股份有限公司 | Circular enzyme detection method for detecting CG (cholyglycine) |
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Open date: 20090624 |