CN101324630A - Method for determining ethyl hydrate concentration ethyl hydrate diagnosis reagent kit - Google Patents

Method for determining ethyl hydrate concentration ethyl hydrate diagnosis reagent kit Download PDF

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Publication number
CN101324630A
CN101324630A CNA200710023209XA CN200710023209A CN101324630A CN 101324630 A CN101324630 A CN 101324630A CN A200710023209X A CNA200710023209X A CN A200710023209XA CN 200710023209 A CN200710023209 A CN 200710023209A CN 101324630 A CN101324630 A CN 101324630A
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CN
China
Prior art keywords
reagent
coenzyme
stabilizing agent
aldehyde dehydrogenase
hydrogen peroxide
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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CNA200710023209XA
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Chinese (zh)
Inventor
王尔中
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou ANJ Biotech Co Ltd
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Suzhou ANJ Biotech Co Ltd
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Publication date
Application filed by Suzhou ANJ Biotech Co Ltd filed Critical Suzhou ANJ Biotech Co Ltd
Priority to CNA200710023209XA priority Critical patent/CN101324630A/en
Publication of CN101324630A publication Critical patent/CN101324630A/en
Pending legal-status Critical Current

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Abstract

The invention relates to a kit for diagnosing/mensurating ethanol by utilizing the technologies of the enzymic colorimetric method 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 mensurating the concentration of the ethanol, and belongs to the technology field of medical/food/environmental inspection and measurement. The main components of the kit include a buffer solution, coenzyme, hydrogen peroxide, catalase, aldehyde dehydrogenase and a stabilizer. Through mixing a sample and the reagent by a certain volume ratio, an enzymatic reaction occurs, 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 ethanol.

Description

Diagnosing/determining alcohol kit and concentration of ethanol assay method
Technical field
The present invention relates to a kind of diagnosing/determining alcohol kit, the invention still further relates to the method for measuring concentration of alcohol simultaneously, belong to medical science/food/environmental test determination techniques field.
Background technology
Ethanol tool habituation, alcohol abuse and alcohol dependence are one of serious social economic and public health problems in world today's scope.In western countries, alcohol correlativity hepatopathy has become the 6th dead reason.In China, over nearly 20 years, along with socio-economic development, the wine consumption rises significantly per capita, and the morbidity rate of alcohol dependence increases day by day.
The method for quantitatively determining of ethanol mainly contains chemical colorimetry in the biological sample, the enzyme terminal colorimetric analysis, and homogeneous EIA method (EIA), vapor-phase chromatography (GC) and expiratory air ethanol are analyzed.Serum infiltration capacity method can be made the ethanol semi-quantitative analysis in addition.
Have the number of chemical colourimetry once to be used for the ethanol quantitative measurement, microdiffusion is wherein more simple and practical a kind of, and the ethanol that trace overflows can make chromic acid be reduced into blue chromium oxide.
According to used reagent enzyme difference, the enzymatic assays of ethanol is divided into two kinds: oxidation of ethanol enzyme process and alcohol dehydrogenase enzyme process, to compare with the oxidation of ethanol enzyme process, and the selectivity height of alcohol dehydrogenase enzyme process can not play catalytic reaction with methyl alcohol and acetone.
It is generally acknowledged that vapor-phase chromatography can be used as reference method, especially most widely used in the high medical jurisprudence alcohol determining of accuracy and precision requirement.The distinct advantages of GC method is special, sensitive, and can analyze multiple volatility alcohols material simultaneously, as ethanol, methyl alcohol, acetone, acetaldehyde and isopropyl alcohol.
The assay method of other ethanol also has: homogeneous enzyme immunoassay analysis (Homogeneous enzymeimmunoassay), infiltration capacity method, expiratory air ethanol analytic approach; It is simple, quick to detect ethanol operation with expiratory air ethanol analyser, uses more in traffic administration department.A kind of according to the infrared Absorption principle design in this quasi-instrument, the blood concentration of alcohol correlativity that concentration of alcohol that this method is measured and enzyme process record is fine.
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, metering/continuous monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for concentration of alcohol, simultaneously, the present invention also will provide in order to realize the diagnosing/determining alcohol kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carry out determining concentration of alcohol on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Method for determining concentration of alcohol principle of the present invention is as follows:
Ethanol+peroxidating Hydroperoxidation hydrogen enzymeAcetaldehyde+water
Acetaldehyde+coenzyme+water Aldehyde dehydrogenaseAcetate+reduced coenzyme
This method application of catalase (catalase; EC 1.11.1.6) enzyme (idol) connection aldehyde dehydrogenase (Aldehyde dehydrogenase; EC 1.2.1.3; EC 1.2.1.4; EC 1.2.1.5) enzyme ' s reaction speeding colourimetry/end-point method.Hydrogen peroxidase enzymolysis ethanol synthesis produces acetaldehyde, the effect of uniting aldehyde dehydrogenase again by (idol), 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 degree/speed that reduced coenzyme rises in 340nm place absorbance, by measuring degree/speed that 340nm place absorbance rises, can calculate the concentration of ethanol size.
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 diagnosing/determining alcohol kit of the present invention of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Hydrogen peroxide 3mmol/L
Hydrogen peroxidase 10000U/L
Aldehyde dehydrogenase 10000U/L
Diagnosing/determining alcohol kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, hydrogen peroxide, hydrogen peroxidase, aldehyde 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, hydrogen peroxide.
Reagent 2
Damping fluid, stabilizing agent, hydrogen peroxidase, aldehyde dehydrogenase.
Coenzyme, hydrogen peroxide, hydrogen peroxidase, the position of aldehyde 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, coenzyme, hydrogen peroxide.
Reagent 2
Damping fluid, stabilizing agent, aldehyde dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, hydrogen peroxidase.
Coenzyme, hydrogen peroxide, hydrogen peroxidase, the position of aldehyde 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 concentration of alcohol, 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 diagnosing/determining alcohol reagent of present embodiment is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Hydrogen peroxide 3mmol/L
Hydrogen peroxidase 10000U/L
Aldehyde dehydrogenase 10000U/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 ethanol 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 ethanol size.
Embodiment two
The diagnosing/determining alcohol reagent of present embodiment is double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Hydrogen peroxide 3mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Hydrogen peroxidase 10000U/L
Aldehyde dehydrogenase 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≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested ethanol 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 ethanol size.
Embodiment three
The diagnosing/determining alcohol reagent of present embodiment is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Hydrogen peroxide 3mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Aldehyde dehydrogenase 10000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Hydrogen peroxidase 10000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring concentration of alcohol, 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 ethanol 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 ethanol 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 (6)

1. the concentration of ethanol assay method of enzymic colorimetric and enzyme-linked method, its method principle is as follows:
Ethanol+hydrogen peroxide Hydrogen peroxidaseAcetaldehyde+water
Acetaldehyde+coenzyme+water Aldehyde dehydrogenaseAcetate+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 concentration of ethanol size measurement result.
2. diagnosing/determining alcohol kit, principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
DPN diphosphopyridine nucleotide---6mmol/L
Hydrogen peroxide 1---6mmol/L
Hydrogen peroxidase 1000---80000U/L
Aldehyde 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 diagnosing/determining alcohol kit of claim 2, it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, coenzyme, hydrogen peroxide, hydrogen peroxidase, aldehyde dehydrogenase.
4. according to the described diagnosing/determining alcohol kit of claim 2, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, coenzyme, hydrogen peroxide, hydrogen peroxidase, aldehyde dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, hydrogen peroxide; Reagent 2 is made up of damping fluid, stabilizing agent, hydrogen peroxidase, aldehyde dehydrogenase.Coenzyme, hydrogen peroxide, hydrogen peroxidase, the position of aldehyde dehydrogenase in reagent 1 or reagent 2 can not limit.
5. according to the described diagnosing/determining alcohol kit of claim 2, it is characterized in that: form multi-agent reagent by damping fluid, stabilizing agent, coenzyme, hydrogen peroxide, hydrogen peroxidase, aldehyde dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, hydrogen peroxide; Reagent 2 is made up of damping fluid, stabilizing agent, aldehyde dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, hydrogen peroxidase.Coenzyme, hydrogen peroxide, hydrogen peroxidase, the position of aldehyde dehydrogenase in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described diagnosing/determining alcohol 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 (PropyleneGlycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
CNA200710023209XA 2007-06-13 2007-06-13 Method for determining ethyl hydrate concentration ethyl hydrate diagnosis reagent kit Pending CN101324630A (en)

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CN101324630A true CN101324630A (en) 2008-12-17

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3214180A1 (en) * 2016-03-01 2017-09-06 Sani-Marc Inc. Methods, compositions and kits for determining cleanness of a surface
US10920263B2 (en) 2016-03-01 2021-02-16 Sani-Marc Inc. Methods, compositions and kits for determining cleanness of a surface

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3331752A (en) * 1966-06-07 1967-07-18 Ortho Pharma Corp Determination of dehydrogenase
JPS6191570A (en) * 1984-10-12 1986-05-09 Amano Pharmaceut Co Ltd Assay of reducing type nicotinamide adenine dinucleotide and reducing type nicotinamide adenine dinucleotide phosphate
CN1746316A (en) * 2004-09-08 2006-03-15 王尔中 Determination of adenosine deaminase activity and diagnostic kit of adenosine deaminase
CN1749756A (en) * 2004-09-14 2006-03-22 王尔中 Method for detecting blood ammonia content and blood ammonia diagnostic reagent kit
CN1749411A (en) * 2004-09-14 2006-03-22 王尔中 Method for measuring 5'-nucleotidase activity and diagnostic kit of 5'-nucleotidase
CN1757751A (en) * 2004-10-10 2006-04-12 王尔中 Determination method of creatnine content and reagent box for diagnosing creatnine

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3331752A (en) * 1966-06-07 1967-07-18 Ortho Pharma Corp Determination of dehydrogenase
JPS6191570A (en) * 1984-10-12 1986-05-09 Amano Pharmaceut Co Ltd Assay of reducing type nicotinamide adenine dinucleotide and reducing type nicotinamide adenine dinucleotide phosphate
CN1746316A (en) * 2004-09-08 2006-03-15 王尔中 Determination of adenosine deaminase activity and diagnostic kit of adenosine deaminase
CN1749756A (en) * 2004-09-14 2006-03-22 王尔中 Method for detecting blood ammonia content and blood ammonia diagnostic reagent kit
CN1749411A (en) * 2004-09-14 2006-03-22 王尔中 Method for measuring 5'-nucleotidase activity and diagnostic kit of 5'-nucleotidase
CN1757751A (en) * 2004-10-10 2006-04-12 王尔中 Determination method of creatnine content and reagent box for diagnosing creatnine

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3214180A1 (en) * 2016-03-01 2017-09-06 Sani-Marc Inc. Methods, compositions and kits for determining cleanness of a surface
US10920263B2 (en) 2016-03-01 2021-02-16 Sani-Marc Inc. Methods, compositions and kits for determining cleanness of a surface

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Application publication date: 20081217