CN101324555A - D-lactic acid diagnosis/determination reagent kit and method for determining D-lactic acid concentration - Google Patents
D-lactic acid diagnosis/determination reagent kit and method for determining D-lactic acid concentration Download PDFInfo
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- CN101324555A CN101324555A CNA2007100232244A CN200710023224A CN101324555A CN 101324555 A CN101324555 A CN 101324555A CN A2007100232244 A CNA2007100232244 A CN A2007100232244A CN 200710023224 A CN200710023224 A CN 200710023224A CN 101324555 A CN101324555 A CN 101324555A
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- lactic acid
- reagent
- coenzyme
- stabilizing agent
- lactic
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Abstract
The invention relates to a kit for diagnosing/mensurating D-lactic acid by utilizing the technology of the enzymic colorimetric method based on the specificity of the D-lactic acid. The invention further relates to a method, a principle and the composition and the components of a reagent for mensurating the concentration of the D-lactic acid, and belongs to the technology field of medical/food inspection and measurement. The main components of the kit include a buffer solution, coenzyme, D-lactate dehydrogenase 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 340nm of the dominant wavelength is detected, thereby mensurating the concentration of the D-lactic acid.
Description
Technical field
The present invention relates to a kind of D-lactic acid diagnosis/determination kit, the invention still further relates to the method for measuring the D-lactic acid concn simultaneously, belong to medical science/Food Inspection determination techniques field.
Background technology
L-lactic acid is that people extremely pay close attention to and develop organic acid faster in recent years.L-lactic acid has in multi-field application prospects such as food, feed, medicine, plastics, feed, agricultural chemicals, daily-use chemical industry, papermaking and electronics industries simultaneously.
L-lactic acid is the metabolic end-products of most of biosomes.Only minority microorganism (routine genus lactubacillus, Leuconostoc) can form D-lactic acid.Exactissima diligentia detects lactate when producing sour milk, whether produces L-lactic acid with the assessment microorganism, and D-lactic acid or the two have concurrently.If there is D-lactic acid to exist, then may be that microorganism has been caused pollution.
The unusual rising of lactate concentration and multiple disease such as diseases such as diabetes, acidosis have confidential relation.L (+)-Lactate is the main intermediate product of lactate metabolism in the human body, and is present in the blood.D (-)-Lactate also exists, but content is very low, has only the 1-5% of L (+)-Lactate.
The test of blood lactic acid is as the means of a monitoring, monitoring training athlete ability, the significant data of blood lactic acid value as aspects such as measurement sportsman aerobic endurance level, anaerobic exercise ability, physical fatigue recoveries, reference during for the arrangement training.
Enzymatic analysis is the analytical approach 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.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of utilization and have the specific enzymic colorimetric of D-lactic acid (Enzymatic Colorimetric Method) technology, metering/continuous monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for D-lactic acid concn, simultaneously, the present invention also will provide in order to realize the D-lactic acid diagnosis/determination kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carrying out the D-lactic acid concn on the automatic clinical chemistry analyzer measures, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
D-lactic acid concn assay method principle of the present invention is as follows:
D-lactic acid+coenzyme
The D-lactic dehydrogenasePyruvic acid+reduced coenzyme
This method is used D-lactic dehydrogenase (D-lactic acid dehydrogenase; EC 1.1.1.28) enzyme ' s reaction speeding colourimetry/end-point method.D-lactic dehydrogenase enzymolysis D-lactic acid, and with coenzyme (not having absorption peak) reduction becoming reduced coenzyme (absorption peak being arranged) at the 340nm place 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 D-concentration of lactic acid size.
Experiment shows, takes all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, and no matter be single agent, two agent, the D-lactic acid diagnosis/determination kit of the present invention of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
D-lactic dehydrogenase 12000U/L
D-lactic acid diagnosis/determination kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, D-lactic 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.
Reagent 2
Damping fluid, stabilizing agent, D-lactic dehydrogenase.
Coenzyme, the position of D-lactic 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.
No matter be single agent, two agent, the present invention measures the method for D-lactic acid concn, 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
The D-lactic acid diagnosing/determining reagent of present embodiment is single reagent, comprising:
Glycocoll-sodium hydrate buffer solution 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
D-lactic dehydrogenase 12000U/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 D-lactic 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 D-concentration of lactic acid size.
Embodiment two
The D-lactic acid diagnosing/determining reagent of present embodiment is double reagent, comprising:
Reagent 1
Glycocoll-sodium hydrate buffer solution 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Reagent 2
Glycocoll-sodium hydrate buffer solution 100mmol/L
Stabilizing agent 500mmol/L
D-lactic dehydrogenase 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 D-lactic 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 D-concentration of lactic acid size.
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 (5)
1. one kind has the specific enzymic colorimetric D-of D-lactic acid lactic acid concn assay method, and its method principle is as follows:
D-lactic acid+coenzyme
The D-lactic dehydrogenasePyruvic 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 D-concentration of lactic acid size measurement result.
2. D-lactic acid diagnosis/determination kit, principal ingredient comprises:
Damping fluid 20-500mmol/L
Stabilizing agent 1-4000mmol/L
DPN diphosphopyridine nucleotide-6mmol/L
D-lactic dehydrogenase 10 00-80000U/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 D-lactic acid of claim 2 diagnosis/determination kit, it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, coenzyme, D-lactic dehydrogenase.
4. according to the described D-lactic acid of claim 2 diagnosis/determination kit, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, coenzyme, D-lactic dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme; Reagent 2 is made up of damping fluid, stabilizing agent, D-lactic dehydrogenase.Coenzyme, the position of D-lactic dehydrogenase in reagent 1 or reagent 2 can not limit.
5. according to the described D-lactic acid of claim 2 diagnosis/determination 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 |
|---|---|---|---|
| CNA2007100232244A CN101324555A (en) | 2007-06-13 | 2007-06-13 | D-lactic acid diagnosis/determination reagent kit and method for determining D-lactic acid concentration |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100232244A CN101324555A (en) | 2007-06-13 | 2007-06-13 | D-lactic acid diagnosis/determination reagent kit and method for determining D-lactic acid concentration |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101324555A true CN101324555A (en) | 2008-12-17 |
Family
ID=40188179
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007100232244A Pending CN101324555A (en) | 2007-06-13 | 2007-06-13 | D-lactic acid diagnosis/determination reagent kit and method for determining D-lactic acid concentration |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101324555A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507915A (en) * | 2011-11-07 | 2012-06-20 | 宁波美康生物科技股份有限公司 | Stable liquid kit for measuring lactic acid |
| WO2024051646A1 (en) * | 2022-09-06 | 2024-03-14 | 华东理工大学 | Method and kit for diagnosing midd |
-
2007
- 2007-06-13 CN CNA2007100232244A patent/CN101324555A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507915A (en) * | 2011-11-07 | 2012-06-20 | 宁波美康生物科技股份有限公司 | Stable liquid kit for measuring lactic acid |
| CN102507915B (en) * | 2011-11-07 | 2014-01-01 | 宁波美康生物科技股份有限公司 | Stable liquid kit for measuring lactic acid |
| WO2024051646A1 (en) * | 2022-09-06 | 2024-03-14 | 华东理工大学 | Method and kit for diagnosing midd |
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Open date: 20081217 |