CN101329264A - Sucrase diagnosis / determination reagent kit and method for determining sucrase active concentration - Google Patents
Sucrase diagnosis / determination reagent kit and method for determining sucrase active concentration Download PDFInfo
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- CN101329264A CN101329264A CNA2007100247339A CN200710024733A CN101329264A CN 101329264 A CN101329264 A CN 101329264A CN A2007100247339 A CNA2007100247339 A CN A2007100247339A CN 200710024733 A CN200710024733 A CN 200710024733A CN 101329264 A CN101329264 A CN 101329264A
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- invertase
- coenzyme
- stabilizing agent
- sucrose
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Abstract
The invention relates to an invertase diagnosis/determination kit which uses the techniques of an enzyme colorimetry method and an enzyme-linked method; meanwhile, the invention also relates to a method principle used for determining invertase activity consistency, reagent composition and component, belonging to the field of medical /food detection determination technique. The main components of the kit of the invention comprise buffer solution, coenzyme, saccharose, fructose dehydrogenase and stabilizer; samples and reagents are mixed according to a certain volume proportion so as to generate a series of enzymatic reactions; subsequently, reaction matters are arranged under an ultraviolet/visible light analyser so as to detect the degree/speed of the absorbency increment of the main wavelength at the 340nm position, thus calculating the activity consistency of the invertase.
Description
Technical field
The present invention relates to a kind of invertase diagnosis/mensuration kit, the invention still further relates to the method for measuring sucrase active concentration simultaneously, belong to medical science/Food Inspection determination techniques field.
Background technology
The function of invertase (sucrase) is called invertase (invertase) or invertase (saccharase) again for sucrose hydrolysis is become D-fructose and D-glucose.Sucrase-isomaltase deficiency, in a single day the known mankind lack wherein a kind of enzyme, will produce the hereditary disease to the disaccharides impatience, might they be to coexist on the protein, belong to single-gene control.Invertase plays a part crucial in the higher plant Sucrose Metabolism.
The detection method of invertase has: (1) Nelson method, its principle is: invertase can be glucose and fructose with sucrose hydrolysis, and glucose can be with Cu in alkaline solution
2Reduction; Arsenic molybdic acid reagent and cuprous oxide generate blue compound (arsenic molybdenum blue), and the optical density that is proportional to concentration of reduced sugar is arranged under the 510nm wavelength, thereby determine the concentration of invertase.(2) salicylic acid method: invertase can make sucrose hydrolysis become glucose and fructose, glucose and 3, the 5-dinitrosalicylic acid is reduced into henna amino-compound after the heat altogether, and in the finite concentration scope, the amount of glucose and brownish red material color depth degree are proportional.(3) with the colorimetric estimation of ortho-aminotoluene reagent.
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, continuous monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for sucrase active concentration, simultaneously, the present invention also will provide in order to realize the invertase diagnosis/mensuration kit of this method, adopt this kit not only can be ultraviolet analyser or half, carry out the sucrase active concentration determination on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Sucrase active method for measurement of concentration principle of the present invention is as follows:
Sucrose
InvertaseD-glucose+D-fructose
D-fructose+coenzyme
Fructose dehydrogenaseDehydrogenation fructose+reduced coenzyme
This method is used invertase (sucrase; EC 3.2.1.48) coupling Fructose dehydrogenase (Fructosedehydrogenase; EC 1.1.1.124; EC 1.1.99.11) enzymatic reaction continuous monitoring method.The reaction of invertase enzymolysis sucrose produces fructose, effect by the coupling Fructose 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 the speed that reduced coenzyme rises in 340nm place absorbance, by measuring the speed that 340nm place absorbance rises, can calculate the active concentration size of invertase enzyme.
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 invertase diagnosis/mensuration reagent of the present invention of following composition relation is ideal comparatively:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Sucrose 30mmol/L
Fructose dehydrogenase 9000U/L
Invertase diagnosis/mensuration kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, Fructose dehydrogenase, sucrose.
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, sucrose.
Reagent 2
Damping fluid, stabilizing agent, Fructose dehydrogenase.
Coenzyme, Fructose dehydrogenase, the position of sucrose 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.
Reagent 2
Damping fluid, sucrose.
Reagent 3
Damping fluid, stabilizing agent, Fructose dehydrogenase.
Coenzyme, Fructose dehydrogenase, the position of sucrose 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 sucrase active 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
Invertase 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
Sucrose 30mmol/L
Fructose dehydrogenase 9000U/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 invertase sample and reagent is 1/25, the Direction of Reaction is positive reaction (reaction of rising), 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 rises, thereby calculates the active concentration size of invertase.
Embodiment two
Invertase 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
Sucrose 30mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Fructose dehydrogenase 9000U/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 invertase sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is positive reaction (reaction of rising), 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 rises, thereby calculates the active concentration size of invertase.
Embodiment three
Invertase 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
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Sucrose 30mmol/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Fructose dehydrogenase 9000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring sucrase active 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 invertase sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, and the Direction of Reaction is positive reaction (reaction of rising), 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 rises, thereby calculates the active concentration size of invertase.
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. sucrase active method for measurement of concentration that utilizes enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:
Sucrose
InvertaseD-glucose+D-fructose
D-fructose+coenzyme
Fructose dehydrogenaseDehydrogenation fructose+reduced coenzyme
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the speed that predominant wavelength 340nm absorbance rises, calculate the active concentration size measurement result of invertase.
2. invertase diagnosis/mensuration kit, principal ingredient comprises:
Damping fluid 20-500mmol/L
Stabilizing agent 1-4000mmol/L
DPN diphosphopyridine nucleotide-6mmol/L
Sucrose 1-50mmol/L
Fructose 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 invertase diagnosis/mensuration of claim 2 kit, it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, coenzyme, Fructose dehydrogenase, sucrose.
4. according to the described invertase diagnosis/mensuration of claim 2 kit, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, coenzyme, Fructose dehydrogenase, sucrose; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, sucrose; Reagent 2 is made up of damping fluid, stabilizing agent, Fructose dehydrogenase.Coenzyme, Fructose dehydrogenase, the position of sucrose in reagent 1 or reagent 2 can not limit.
5. according to the described invertase diagnosis/mensuration of claim 2 kit, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, coenzyme, Fructose dehydrogenase, sucrose; Reagent 1 is by damping fluid, stabilizing agent, coenzyme, form; Reagent 2 is made up of damping fluid, stabilizing agent, sucrose; Reagent 3 is made up of damping fluid, stabilizing agent, Fructose dehydrogenase.Coenzyme, Fructose dehydrogenase, the position of sucrose in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described invertase 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 |
|---|---|---|---|
| CNA2007100247339A CN101329264A (en) | 2007-06-21 | 2007-06-21 | Sucrase diagnosis / determination reagent kit and method for determining sucrase active concentration |
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| CNA2007100247339A CN101329264A (en) | 2007-06-21 | 2007-06-21 | Sucrase diagnosis / determination reagent kit and method for determining sucrase active concentration |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102539712A (en) * | 2010-12-13 | 2012-07-04 | 苏州艾杰生物科技有限公司 | Method for determining ammonia (ammonia ion) and ammonia (ammonia ion) diagnosis/determination kit |
| CN102741694A (en) * | 2009-12-16 | 2012-10-17 | 霍夫曼-拉罗奇有限公司 | Detecting the decomposition of enzymes in a test element by means of controlled release of protected analyte |
-
2007
- 2007-06-21 CN CNA2007100247339A patent/CN101329264A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102741694A (en) * | 2009-12-16 | 2012-10-17 | 霍夫曼-拉罗奇有限公司 | Detecting the decomposition of enzymes in a test element by means of controlled release of protected analyte |
| CN102741694B (en) * | 2009-12-16 | 2014-12-24 | 霍夫曼-拉罗奇有限公司 | Detecting the decomposition of enzymes in a test element by means of controlled release of protected analyte |
| US10260085B2 (en) | 2009-12-16 | 2019-04-16 | Roche Diabetes Care, Inc. | Detecting the decomposition of enzymes in a test element by means of controlled release of a protected analyte |
| CN102539712A (en) * | 2010-12-13 | 2012-07-04 | 苏州艾杰生物科技有限公司 | Method for determining ammonia (ammonia ion) and ammonia (ammonia ion) diagnosis/determination kit |
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