CN101750369A - Ethanol diagnostic/test reagent (kit) and measuring method of ethanol concentration - Google Patents
Ethanol diagnostic/test reagent (kit) and measuring method of ethanol concentration Download PDFInfo
- Publication number
- CN101750369A CN101750369A CN200810244249A CN200810244249A CN101750369A CN 101750369 A CN101750369 A CN 101750369A CN 200810244249 A CN200810244249 A CN 200810244249A CN 200810244249 A CN200810244249 A CN 200810244249A CN 101750369 A CN101750369 A CN 101750369A
- Authority
- CN
- China
- Prior art keywords
- reagent
- coenzyme
- alcohol
- oxidase
- enzyme
- Prior art date
- 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
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to an ethanol diagnostic/test reagent (kit) utilizing an enzyme multiplied method, an enzyme colorimetric method and an enzyme coupling method, and the invention also relates to a measuring method of ethanol concentration, and components and ingredients of the test reagent, which belongs to the technical scheme of medical/food/environmental test measurement. The main ingredients of the test reagent (kit) of the invention comprise buffer solution, coenzyme, coenzyme A, alcohol oxidizing enzyme, acetaldehyde dehydrogenase, NAD (P) H oxidizing enzyme and stabilizing agent; a sampler is mixed with the test reagent according to a certain volume ration to have a series of enzymatic reaction, and then the reactant is put under a ultraviolet/visible light analyzer to detect the rising degree of the absorbance on the 340nm position of the main wave length, thus measuring and calculating the concentration of the ethanol.
Description
Technical field
The present invention relates to a kind of diagnosing/determining alcohol reagent (box), 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 enzyme multiplication method (Enzymatic DoublingMethod) that utilizes, enzymic colorimetric (Enzymatic Colorimetric Method) and enzyme (even) united method (CoupleReaction) technology, metering 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 reagent (box) 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 of the present invention is as follows:
Ethanol+oxygen
Alcohol oxidase enzymeAcetaldehyde+hydrogen peroxide
Acetaldehyde+coacetylase+coenzyme
Acetaldehyde dehydrogenaseAcetyl-coacetylase+reduced coenzyme
Peroxidating oxygen+coenzyme
NAD (P) H oxidaseReduced coenzyme+oxygen
This method is used alcohol oxidase enzyme (alcohol oxidase; EC 1.1.3.13) enzyme (idol) connection acetaldehyde dehydrogenase (acetaldehyde dehydrogenase; EC 1.2.1.10), NAD (P) H oxidase (NAD (P) Hoxidase; EC 1.6.3.1) enzymatic reaction colorimetric end-point method.Alcohol oxidase enzyme enzymolysis ethanol synthesis produces acetaldehyde and hydrogen peroxide, again by (idol) associating acetaldehyde dehydrogenase, the oxidasic effect of NAD (P) H, final secondary is with coenzyme (not having absorption peak at the 340nm place) reduction becoming reduced coenzyme (absorption peak being arranged at the 340nm place), thereby measured the degree that reduced coenzyme rises in 340nm place absorbance, by measuring the degree 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 reagent of the present invention (box) of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Coacetylase 9mmol/L
Alcohol oxidase enzyme 10000U/L
Acetaldehyde dehydrogenase 12000U/L
NAD (P) H oxidase 12000U/L
Diagnosing/determining alcohol reagent of the present invention (box) can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, coacetylase, alcohol oxidase enzyme, acetaldehyde dehydrogenase, NAD (P) H oxidase.
Reagent (box) can be 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, coacetylase.
Reagent 2
Damping fluid, stabilizing agent, alcohol oxidase enzyme, acetaldehyde dehydrogenase, NAD (P) H oxidase.
Coenzyme, coacetylase, alcohol oxidase enzyme, acetaldehyde dehydrogenase, the position of NAD (P) H oxidase in reagent 1 or reagent 2 can not limit.Reagent (box) can be 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, coacetylase.
Reagent 2
Damping fluid, stabilizing agent, acetaldehyde dehydrogenase, NAD (P) H oxidase.
Reagent 3
Damping fluid, stabilizing agent, alcohol oxidase enzyme.
Coenzyme, coacetylase, alcohol oxidase enzyme, acetaldehyde dehydrogenase, the position of NAD (P) H oxidase in reagent 1, reagent 2 or reagent 3 can not limit.Reagent (box) can be 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
Coacetylase 9mmol/L
Alcohol oxidase enzyme 10000U/L
Acetaldehyde dehydrogenase 12000U/L
NAD (P) H oxidase 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 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 the degree 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
Coacetylase 9mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Alcohol oxidase enzyme 10000U/L
Acetaldehyde dehydrogenase 12000U/L
NAD (P) H oxidase 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 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 the degree 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
Coacetylase 9mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Acetaldehyde dehydrogenase 12000U/L
NAD (P) H oxidase 12000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Alcohol oxidase enzyme 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 the degree that predominant wavelength 340nm absorbance rises, thereby calculates the concentration of ethanol size.
The applicant adopts other assay methods 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, experimental results show that: adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully---the blank reagent absorbance changes (Δ A/min)≤0.0005; Absorbance time response curve should be the rising curve until terminal point; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 20mmol/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 4%; The coefficient of variation (CV)≤2% of the precision of reagent test (repeatability); The sensitivity of reagent can reach 0.0060 ± 0.0030 Δ A/mmol/L; Reagent is preserved down at 2-8 ℃, and activity can be stablized 1 year;---the present invention is highly sensitive, degree of accuracy good, the linear range broadness, and stationary phase is long, is enough to easy to utilize.
Claims (6)
1. the concentration of ethanol assay method of an enzyme multiplication method, enzymic colorimetric and enzyme-linked method, its method is as follows:
Ethanol+oxygen
Alcohol oxidase enzymeAcetaldehyde+hydrogen peroxide
Acetaldehyde+coacetylase+coenzyme
Acetaldehyde dehydrogenaseAcetyl-coacetylase+reduced coenzyme
Hydrogen peroxide+coenzyme
NAD (P) H oxidaseReduced coenzyme+oxygen
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the degree that predominant wavelength 340nm absorbance rises, calculate concentration of ethanol size measurement result.
2. a diagnosing/determining alcohol reagent (box), principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
DPN diphosphopyridine nucleotide---6mmol/L
Alcohol oxidase enzyme 1000---80000U/L
Acetaldehyde dehydrogenase 1000---80000U/L
NAD (P) H oxidase 1000---80000U/L
Coacetylase 1---50mmol/L
The concentration of reagent composition not necessarily is only limited to above-mentioned scope; Effect is better in this scope, and outside this scope, reagent still can reagentia.
It is characterized in that: reagent (box) can be 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 reagent of claim 2 (box), it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, coenzyme, coacetylase, alcohol oxidase enzyme, acetaldehyde dehydrogenase, NAD (P) H oxidase.
4. according to the described diagnosing/determining alcohol reagent of claim 2 (box), it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, coenzyme, coacetylase, alcohol oxidase enzyme, acetaldehyde dehydrogenase, NAD (P) H oxidase; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, coacetylase; Reagent 2 is made up of damping fluid, stabilizing agent, alcohol oxidase enzyme, acetaldehyde dehydrogenase, NAD (P) H oxidase.Coenzyme, coacetylase, alcohol oxidase enzyme, acetaldehyde dehydrogenase, the position of NAD (P) H oxidase in reagent 1 or reagent 2 can not limit.
5. according to the described diagnosing/determining alcohol reagent of claim 2 (box), it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, coenzyme, coacetylase, alcohol oxidase enzyme, acetaldehyde dehydrogenase, NAD (P) H oxidase; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, coacetylase; Reagent 2 is made up of damping fluid, stabilizing agent, acetaldehyde dehydrogenase, NAD (P) H oxidase; Reagent 3 is made up of damping fluid, stabilizing agent, alcohol oxidase enzyme.Coenzyme, coacetylase, alcohol oxidase enzyme, acetaldehyde dehydrogenase, the position of NAD (P) H oxidase in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described diagnosing/determining alcohol reagent of claim 2 (box), it is characterized in that: also comprise stabilizing agent 1-4000mmol/L or 0.1%-100% volume ratio.Described stabilizing agent is: ammonium sulfate (AmmoniaSulfate), 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 |
|---|---|---|---|
| CN200810244249A CN101750369A (en) | 2008-12-10 | 2008-12-10 | Ethanol diagnostic/test reagent (kit) and measuring method of ethanol concentration |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810244249A CN101750369A (en) | 2008-12-10 | 2008-12-10 | Ethanol diagnostic/test reagent (kit) and measuring method of ethanol concentration |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101750369A true CN101750369A (en) | 2010-06-23 |
Family
ID=42477669
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810244249A Pending CN101750369A (en) | 2008-12-10 | 2008-12-10 | Ethanol diagnostic/test reagent (kit) and measuring method of ethanol concentration |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101750369A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110057640A (en) * | 2019-04-30 | 2019-07-26 | 山东博科生物产业有限公司 | Anti-interference, stable compound calibration object and preparation method containing 33 indexs |
-
2008
- 2008-12-10 CN CN200810244249A patent/CN101750369A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110057640A (en) * | 2019-04-30 | 2019-07-26 | 山东博科生物产业有限公司 | Anti-interference, stable compound calibration object and preparation method containing 33 indexs |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101169448A (en) | Ethanol diagnosis/determination reagent kit and ethanol concentration determination method | |
| CN101762542A (en) | Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol | |
| CN101324630A (en) | Method for determining ethyl hydrate concentration ethyl hydrate diagnosis reagent kit | |
| CN101324628A (en) | Method for determining ethanol concentration and ethanol diagnosis/determination reagent kit | |
| CN101750369A (en) | Ethanol diagnostic/test reagent (kit) and measuring method of ethanol concentration | |
| CN101762538A (en) | Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol | |
| CN101762541A (en) | Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol | |
| CN101762543A (en) | Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol | |
| CN101762481A (en) | Monoamine oxidase diagnostic reagent (kit) and activity concentration determining method of monoamine oxidase | |
| CN101750370A (en) | Ethanol diagnostic/test reagent (kit) and measuring method of ethanol concentration | |
| CN101750375A (en) | Ethanol diagnostic/test reagent (kit) and measuring method of ethanol concentration | |
| CN101750376A (en) | Ethanol diagnostic/test reagent (kit) and measuring method of ethanol concentration | |
| CN101762536A (en) | Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol | |
| CN101750373A (en) | Ethanol diagnostic/test reagent (kit) and measuring method of ethanol concentration | |
| CN101793777A (en) | Reagent (kit) for measuring formaldehyde and method for measuring concentration of formaldehyde | |
| CN101750374A (en) | Ethanol diagnostic/test reagent (kit) and measuring method of ethanol concentration | |
| CN101762540A (en) | Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol | |
| CN101762544A (en) | Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol | |
| CN101762534A (en) | Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol | |
| CN101762537A (en) | Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol | |
| CN101762539A (en) | Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol | |
| CN101169449A (en) | Ethanol diagnosis/determination reagent kit and ethanol concentration determination method | |
| CN101082570A (en) | Method for measuring ethyl hydrate concentration ethyl hydrate diagnose reagent kit | |
| CN101793778A (en) | Reagent (kit) for measuring formaldehyde and method for measuring concentration of formaldehyde | |
| CN101625312A (en) | Manganese ion diagnostic/assay kit and method for assaying concentration of manganese ions |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| DD01 | Delivery of document by public notice |
Addressee: Aijie Biological Science & Technology Co., Ltd., Suzhou City Wang Erzhong Document name: Notification that Application Deemed to be Withdrawn |
|
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20100623 |