CN101173946A - Manganese diagnosis/measuring reagent kit and method for measuring manganese concentration - Google Patents
Manganese diagnosis/measuring reagent kit and method for measuring manganese concentration Download PDFInfo
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- CN101173946A CN101173946A CNA2006100973232A CN200610097323A CN101173946A CN 101173946 A CN101173946 A CN 101173946A CN A2006100973232 A CNA2006100973232 A CN A2006100973232A CN 200610097323 A CN200610097323 A CN 200610097323A CN 101173946 A CN101173946 A CN 101173946A
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- oxaloacetic
- manganese
- arginine
- stabilizing agent
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Abstract
The invention relates to a manganese diagnostic/testing reagent kit using enzymatic colorimetric method and enzymic linkage technology as well as method for testing concentration of manganese as well as composition and content of the reagent, belonging to technical field of medicine/food/environment test. The invention is characterized in that composition in the reagent kit comprises buffer solution, reduction coenzyme, oxaloacetic acid, arginine, oxaloacetic acid decarboxylase, octopine dehydrogenase and stabilizing agent; the sample is mixed with the reagent according to specified ratio to generate a series enzymatic reactions, then the reactant is arranged under a ultraviolet/visible light analyzer to test the decrease speed of absorbance at wavelength of 340nm, thereby the manganese concentration can be tested. The invention has the advantages that the test results can be easily obtained using ultraviolet/visible light analyzer.
Description
Technical field
The present invention relates to a kind of manganese diagnosis/determination kit, the invention still further relates to the method for measuring manganese concentration simultaneously, belong to medical science/food/environmental test determination techniques field.
Background technology
Manganese is indispensable essential composition in human body as a kind of trace element, also is the material poisonous to human body simultaneously.Under state of nature, exist with two valencys, trivalent and tetravalence form, degree of oxidation is low more, and toxicity is high more.Usually manganese has eight kinds of different states of oxidation.The biological significance of manganese is as accessory factor in the human body diversified function to be arranged.Yet, in many enzyme reactions activate, Mn
2+And Mg
2+Can substitute mutually.
Sampling Graphite Furnace Atomic Absorption spectrophotometry commonly used, atomic emissions ICP-OES.It is the best way that neutron excites analysis (NAA), but only can could measure in the good laboratory of some conditions.
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, monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for manganese concentration, simultaneously, the present invention also will provide in order to realize the manganese diagnosis/determination kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carry out measuring manganese concentration on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Method for measuring manganese concentration principle of the present invention is as follows:
Oxaloacetic acid
Oxaloacetic decarboxylase/Mn 2+ Pyruvic acid+carbon dioxide
Pyruvic acid+L-arginine+reduced coenzyme
Octopine dehydrogenaseN2-(D-1-carboxyethyl)-L-arginine+coenzyme+water
Oxaloacetic decarboxylase (the oxaloacetatedecarboxylase that this method application need is manganese ion activated; EC 4.1.1.3) (idol) connection octopine dehydrogenase (octopine dehydrogenase; EC 1.5.1.11) enzyme ' s reaction speeding colourimetry.Oxaloacetic decarboxylase impels oxaloacetic acid generation decarboxylation to produce pyruvic acid, effect by octopine dehydrogenase again, reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into coenzyme (not having absorption peak at the 340nm place) the most at last, thereby measured the speed that reduced coenzyme descends in 340nm place absorbance, by measuring the speed that 340nm place absorbance descends, can calculate the concentration of manganese.
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 manganese diagnosis/determination kit of the present invention of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Oxaloacetic acid 25mmol/L
Arginine 40mmol/L
Oxaloacetic decarboxylase 12000U/L
Octopine dehydrogenase 16000U/L
Manganese diagnosis/determination kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, reduced coenzyme, oxaloacetic acid, arginine, oxaloacetic decarboxylase, octopine 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, reduced coenzyme, oxaloacetic acid, arginine.
Reagent 2
Damping fluid, stabilizing agent, oxaloacetic decarboxylase, octopine dehydrogenase.
Reduced coenzyme, oxaloacetic acid, arginine, oxaloacetic decarboxylase, the position of octopine 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, reduced coenzyme.
Reagent 2
Damping fluid, oxaloacetic acid, arginine.
Reagent 3
Damping fluid, stabilizing agent, oxaloacetic decarboxylase, octopine dehydrogenase.
Reduced coenzyme, oxaloacetic acid, arginine, oxaloacetic decarboxylase, the position of octopine 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 manganese 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 manganese diagnosing/determining reagent of present embodiment is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Oxaloacetic acid 25mmol/L
Arginine 40mmol/L
Oxaloacetic decarboxylase 12000U/L
Octopine 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 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested manganese sample and reagent is 1/25, the Direction of Reaction is negative reaction (reaction descends), 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 manganese.
Embodiment two
The manganese diagnosing/determining 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
Oxaloacetic acid 20mmol/L
Arginine 30mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Oxaloacetic decarboxylase 16000U/L
Octopine dehydrogenase 24000U/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 manganese 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 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 manganese.
Embodiment three
The manganese diagnosing/determining 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
Arginine 30mmol/L
Oxaloacetic acid 40mmol/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Oxaloacetic decarboxylase 24000U/L
Octopine dehydrogenase 36000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring manganese 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 manganese sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, the Direction of Reaction is negative reaction (reaction descends), 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 manganese.
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. method for measuring manganese concentration that utilizes enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:
Oxaloacetic acid oxaloacetic decarboxylase/Mn
2+Pyruvic acid+carbon dioxide
Pyruvic acid+L-arginine+reduced coenzyme
Octopine dehydrogenase
N2-(D-1-carboxyethyl)-L-arginine+coenzyme+water
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 concentration measurement result of manganese.
2. manganese diagnosis/determination kit, principal ingredient comprises:
Damping fluid 20-500mmol/L
Stabilizing agent 1-50mmol/L
Reduced coenzyme 0.1-0.35mmol/L
Oxaloacetic acid 1-50mmol/L
Arginine 1-50mmol/L
Oxaloacetic decarboxylase 1000-80000U/L
Octopine 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.
3. according to the described manganese diagnosis/determination kit of claim 2, it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, oxaloacetic acid, arginine, oxaloacetic decarboxylase, octopine dehydrogenase.
4. according to the described manganese diagnosis/determination kit of claim 2, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, oxaloacetic acid, arginine, oxaloacetic decarboxylase, octopine dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, oxaloacetic acid, arginine; Reagent 2 is made up of damping fluid, stabilizing agent, oxaloacetic decarboxylase, octopine dehydrogenase.Reduced coenzyme, oxaloacetic acid, arginine, oxaloacetic decarboxylase, the position of octopine dehydrogenase in reagent 1 or reagent 2 can not limit.
5. according to the described manganese diagnosis/determination kit of claim 2, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, oxaloacetic acid, arginine, oxaloacetic decarboxylase, octopine dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme; Reagent 2 is made up of damping fluid, oxaloacetic acid, arginine; Reagent 3 is made up of damping fluid, stabilizing agent, oxaloacetic decarboxylase, octopine dehydrogenase.Reduced coenzyme, oxaloacetic acid, arginine, oxaloacetic decarboxylase, the position of octopine dehydrogenase in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described manganese diagnosis/determination 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 (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100973232A CN101173946A (en) | 2006-10-30 | 2006-10-30 | Manganese diagnosis/measuring reagent kit and method for measuring manganese concentration |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100973232A CN101173946A (en) | 2006-10-30 | 2006-10-30 | Manganese diagnosis/measuring reagent kit and method for measuring manganese concentration |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101173946A true CN101173946A (en) | 2008-05-07 |
Family
ID=39422580
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2006100973232A Pending CN101173946A (en) | 2006-10-30 | 2006-10-30 | Manganese diagnosis/measuring reagent kit and method for measuring manganese concentration |
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|---|---|
| CN (1) | CN101173946A (en) |
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2006
- 2006-10-30 CN CNA2006100973232A patent/CN101173946A/en active Pending
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Open date: 20080507 |