CN101173938A - Inorganic phosphorus diagnosis/measuring reagent kit and method for measuring inorganic phosphorus concentration - Google Patents
Inorganic phosphorus diagnosis/measuring reagent kit and method for measuring inorganic phosphorus concentration Download PDFInfo
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- CN101173938A CN101173938A CNA2006100973158A CN200610097315A CN101173938A CN 101173938 A CN101173938 A CN 101173938A CN A2006100973158 A CNA2006100973158 A CN A2006100973158A CN 200610097315 A CN200610097315 A CN 200610097315A CN 101173938 A CN101173938 A CN 101173938A
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- glutamine
- stabilizing agent
- reduced coenzyme
- adenosine diphosphate
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Abstract
The invention relates to a diagnosing and determining reagent box for inorganic phosphorus by use of Enzymatic Recycling Method, Enzymatic Colorimetric Method and Enzymic linkage technology, and meanwhile relates to the principle for determining the concentration of inorganic phosphorus, the compositions and components of the reagent, belonging to the technical field of medicine/ foodstuff/ environment testing and determining. The main components of the reagent box comprise buffer solution, adenosine diphosphate, glutamine, Alpha-ketoglutarate, glutamine synthetase, glutamic acid dehydrogenase, glutamic acid oxidase, reduced coenzyme and stabilizer. By mixing the sample and reagent according to specific volume ratio, a series of enzymatic (or enzyme-coupled) reactions will occur; after that, the reactants are placed under an ultraviolet/ visible light analyzer to detect the speed at which the absorbance reduces at the main wavelength 340 nm, and thus the concentration of the inorganic phosphorus can be derived. The invention has the advantages of acquiring the required determination results by means of an ultraviolet/visible light analyzer, and facilitating the popularization and application.
Description
Technical field
The present invention relates to a kind of Phos diagnosis/mensuration kit, the invention still further relates to the assay method of measuring inorganic phosphorus concentration simultaneously, belong to medical science/food/environmental test determination techniques field.
Background technology
87% of phosphorus all is present in the bone in the human body, and phosphorus in the blood and the phosphorus in the bone keep mobile equilibrium, and certain relation is arranged between calcium, the phosphorus concentration in the blood, and normal person's blood calcium raises then that serium inorganic phosphorus reduces, and vice versa.The product of [calcium], [phosphorus] concentration remains on certain limit in the blood plasma; Approximately the product of per 100 milliliters of plasma calcium phosphorus concentrations is 35---40.When the two product greater than 40 the time, calcium and phosphorus are deposited on bone tissue with the bone salts form,, metastatic calcification can take place at＞70 o'clock.When product＜35, then will hinder the calcification of bone tissue, even bone salts is dissolved again, influence osteogenic action, cause rickets or osteomalacia.Blood plasma [Ca], [Pi's] is constant, mainly is subjected to vitamin D, the adjusting of parathyroid hormone and calcitonin.
Because the phosphorus of human body can't directly be measured at present,, the detection of Phos analyzes phosphate anion (H so being actually
2PO
4 1-, HPO
4 2-).Method commonly used has the phosphomolybdic acid reducing process, non-reduced method, dye binding method, enzyme process, isotope dilution mass spectrometry, atomic absorption spectrophotometry and flow injection analysis etc.Decisive method is an isotope dilution mass spectrometry, and the conventional method of the medium experimental determination Phos that WHO recommends is a colourimetry.
The phosphomolybdic acid reducing process has or not proteinology filtrate method and non-deproteinate method again, and the latter needs to add non-ionics to avoid muddy in reagent.Used reductive agent and kinds of surfactants are a lot, and the more stable reductive agent of performance has ferrous sulphate, iron ammonium sulfate, hydrazine sulfate, Mitouer etc.Surfactant is then the most desirable with polyethylene glycol groups phenyl ether (TritonX-100).The non-reduced method of phosphomolybdic acid (direct ultraviolet method) is directly measured the phosphomolybdic acid complex poly compounds at 340nm or 325nm, and method is easy, is convenient to robotization.But jaundice, haemolysis, the turbid serum of fat has light absorption at 340nm, must do the sample blank, otherwise the result is higher.
The enzymatic assays Phos is a development trend, and its advantage is to be stable in the neutral range of inorganic phosphate compounds under the enzyme effect, can be used for the automated analysis of conventional sample.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of enzyme cycle amplification method (Enzymatic Recycling Method) of utilizing, enzymic colorimetric (Enzymatic ColorimetricMethod) and enzyme-linked method (Couple Reaction) technology, metering reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for inorganic phosphorus concentration, simultaneously, the present invention also will provide in order to realize the Phos diagnosis/mensuration kit of this method, adopt this kit not only can be ultraviolet analyser or half, carry out the mensuration of inorganic phosphorus concentration on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Inorganic phosphorus concentration assay method principle of the present invention is as follows:
Phos+glutamine+adenosine diphosphate
Glutamine synthelaseAmmonium ion+glutamic acid+adenosine triphosphate
Ammonia+α-Tong Wuersuan+reduced coenzyme
Glutamte dehydrogenaseGlutamic acid+coenzyme+water
Glutamic acid+water+oxygen
Dglutamic oxidaseAmmonia+α-Tong Wuersuan+hydrogen peroxide
The enzymatic catalysis that this method is used glutamine synthelase (glutamine synthetase EC 6.3.1.2) produces ammonia.
Glutamte dehydrogenase (glutamate dehydrogenase EC 1.4.1.2, EC 1.4.1.3 or EC 1.4.1.4) and dglutamic oxidase (Glutamate oxidase EC 1.4.3.7, EC 1.4.3.11) are as cyclophorase: the enzymatic catalysis of glutamte dehydrogenase produces glutamic acid; Dglutamic oxidase becomes glutamic acid again ammonia and α-Tong Wuersuan more once more, and it is recycling that ammonia constantly is repeated.
Glutamte dehydrogenase (glutamate dehydrogenase EC 1.4.1.2, EC 1.4.1.3 or EC 1.4.1.4) is as the colour developing enzyme, make reduced coenzyme (absorption peak being arranged) be oxidized into oxidized coenzyme (not having absorption peak) at the 340nm place 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 homocysteine concentration.
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 Phos diagnosis/mensuration kit of the present invention of following composition relation is ideal comparatively:
Damping fluid 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Glutamine synthelase 6000U/L
Glutamte dehydrogenase 20000U/L
Dglutamic oxidase 10000U/L
α-Tong Wuersuan 16mmol/L
Adenosine diphosphate 2mmol/L
Glutamine 4mmol/L
Phos diagnosis/mensuration kit of the present invention can be single agent, comprising: damping fluid, stabilizing agent, adenosine diphosphate, glutamine, 2-oxoglutaric acid ester, glutamine synthelase, glutamte dehydrogenase, dglutamic oxidase, reduced coenzyme.
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, adenosine diphosphate, glutamine, 2-oxoglutaric acid ester, reduced coenzyme.
Reagent 2
Damping fluid, stabilizing agent, glutamine synthelase, glutamte dehydrogenase, dglutamic oxidase.
Adenosine diphosphate, glutamine, 2-oxoglutaric acid ester, glutamine synthelase, glutamte dehydrogenase, dglutamic oxidase, the position of reduced coenzyme 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.
Also above-mentioned single agent reagent can be made into following three doses of reagent:
Reagent 1
Damping fluid, stabilizing agent, adenosine diphosphate, glutamine, 2-oxoglutaric acid ester, reduced coenzyme.
Reagent 2
Damping fluid, stabilizing agent, glutamte dehydrogenase, dglutamic oxidase.
Reagent 3
Damping fluid, stabilizing agent, glutamine synthelase.
Adenosine diphosphate, glutamine, 2-oxoglutaric acid ester, glutamine synthelase, glutamte dehydrogenase, dglutamic oxidase, the position of reduced coenzyme 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 inorganic phosphorus 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
Phos diagnosis/mensuration the reagent of present embodiment is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Glutamine synthelase 6000U/L
Glutamte dehydrogenase 20000U/L
Dglutamic oxidase 10000U/L
α-Tong Wuersuan 16mmol/L
Adenosine diphosphate 2mmol/L
Glutamine 4mmol/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 Phos sample and reagent is 1/25, the Direction of Reaction is negative reaction (reaction descends), detection method is a rate method, and about about 1 minute of time delay is about 2 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 the speed that predominant wavelength 340nm absorbance descends, thereby calculates the size of inorganic phosphorus concentration.
Embodiment two
Phos diagnosis/mensuration the reagent of present embodiment is double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
α-Tong Wuersuan 16mmol/L
Adenosine diphosphate 2mmol/L
Glutamine 4mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Glutamine synthelase 6000U/L
Glutamte dehydrogenase 20000U/L
Dglutamic oxidase 10000U/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 Phos sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is negative reaction (reaction descends), detection method is a rate method, and about about 1 minute of time delay is about 2 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 the speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of Phos.
Embodiment three
Phos diagnosis/mensuration the 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
α-Tong Wuersuan 16mmol/L
Adenosine diphosphate 2mmol/L
Glutamine 4mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Glutamte dehydrogenase 20000U/L
Dglutamic oxidase 10000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Glutamine synthelase 6000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring inorganic phosphorus 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 Phos sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, and the Direction of Reaction is negative reaction (reaction descends), and detection method is a rate method, about about 1 minute of time delay is about 2 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 the speed that predominant wavelength 340nm absorbance descends, thereby calculates the size of inorganic phosphorus concentration.
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, be not subjected in the pollution of allogenic material, easy to utilize.
Claims (6)
1. the inorganic phosphorus concentration assay method of an enzyme cycle amplification method, enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:
Phos+glutamine+adenosine diphosphate
Glutamine synthelase
Ammonium ion+glutamic acid+adenosine triphosphate
Ammonia+α-Tong Wuersuan+reduced coenzyme
Glutamte dehydrogenaseGlutamic acid+
Coenzyme+water
Glutamic acid+water+oxygen dglutamic oxidase ammonia+α-Tong Wuersuan+
Hydrogen peroxide
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the big or small speed that predominant wavelength 340nm absorbance descends, calculate inorganic phosphorus concentration size measurement result.
2. Phos diagnosis/mensuration kit, principal ingredient comprises:
Damping fluid 20-500mmol/L
Stabilizing agent 1-50mmol/L
Reduced coenzyme 0.1-0.35mmol/L
Glutamine synthelase 1000-500000U/L
Dglutamic oxidase 1000-500000U/L
Glutamte dehydrogenase 1000-500000U/L
Adenosine diphosphate 1-12mmol/L
Glutamine 4-20mmol/L
2-oxoglutaric acid ester 4-20mmol/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 Phos diagnosis/mensuration of claim 2 kit, it is characterized in that: form single agent reagent by damping fluid, stabilizing agent, adenosine diphosphate, glutamine, 2-oxoglutaric acid ester, glutamine synthelase (glutamine synthetase EC 6.3.1.2), glutamte dehydrogenase (glutamate dehydrogenase EC 1.4.1.2, EC 1.4.1.3, EC 1.4.1.4), dglutamic oxidase (glutamate oxidase EC 1.4.3.7, EC 1.4.3.11), reduced coenzyme.
4. according to the described Phos diagnosis/mensuration of claim 2 kit, it is characterized in that: form two agent reagent by damping fluid, stabilizing agent, adenosine diphosphate, glutamine, 2-oxoglutaric acid ester, glutamine synthelase (glutamine synthetase EC 6.3.1.2), glutamte dehydrogenase (glutamate dehydrogenase EC 1.4.1.2, EC 1.4.1.3, EC 1.4.1.4), dglutamic oxidase (glutamate oxidase EC 1.4.3.7, EC 1.4.3.11), reduced coenzyme; Reagent 1 is made up of damping fluid, stabilizing agent, adenosine diphosphate, glutamine, 2-oxoglutaric acid ester, reduced coenzyme; Reagent 2 is made up of damping fluid, stabilizing agent, glutamine synthelase, glutamte dehydrogenase, dglutamic oxidase, reduced coenzyme.Adenosine diphosphate, glutamine, 2-oxoglutaric acid ester, glutamine synthelase, glutamte dehydrogenase, dglutamic oxidase, the position of reduced coenzyme in reagent 1 or reagent 2 can not limit.
5. according to the described Phos diagnosis/mensuration of claim 2 kit, it is characterized in that: form multi-agent reagent by damping fluid, stabilizing agent, adenosine diphosphate, glutamine, 2-oxoglutaric acid ester, glutamine synthelase (glutamine synthetase EC 6.3.1.2), glutamte dehydrogenase (glutamate dehydrogenase EC 1.4.1.2, EC 1.4.1.3, EC 1.4.1.4), dglutamic oxidase (glutamate oxidase EC 1.4.3.7, EC 1.4.3.11), reduced coenzyme; Reagent 1 is made up of damping fluid, stabilizing agent, adenosine diphosphate, glutamine, 2-oxoglutaric acid ester, reduced coenzyme; Reagent 2 is made up of damping fluid, stabilizing agent, glutamte dehydrogenase, dglutamic oxidase; Reagent 3 is made up of damping fluid, stabilizing agent glutamine synthelase.Adenosine diphosphate, glutamine, 2-oxoglutaric acid ester, glutamine synthelase, glutamte dehydrogenase, dglutamic oxidase, the position of reduced coenzyme in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described Phos 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 (PropyleneGlycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100973158A CN101173938A (en) | 2006-10-30 | 2006-10-30 | Inorganic phosphorus diagnosis/measuring reagent kit and method for measuring inorganic phosphorus concentration |
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|---|---|---|---|
| CNA2006100973158A CN101173938A (en) | 2006-10-30 | 2006-10-30 | Inorganic phosphorus diagnosis/measuring reagent kit and method for measuring inorganic phosphorus concentration |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102298029A (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 |
| CN102298028A (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 |
| CN102298025A (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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- 2006-10-30 CN CNA2006100973158A patent/CN101173938A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102298029A (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 |
| CN102298028A (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 |
| CN102298025A (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: 20080507 |