CN102495220A - Stable reagent kit of ethanol determination liquid - Google Patents
Stable reagent kit of ethanol determination liquid Download PDFInfo
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- CN102495220A CN102495220A CN2011103472617A CN201110347261A CN102495220A CN 102495220 A CN102495220 A CN 102495220A CN 2011103472617 A CN2011103472617 A CN 2011103472617A CN 201110347261 A CN201110347261 A CN 201110347261A CN 102495220 A CN102495220 A CN 102495220A
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Abstract
The invention discloses a stable reagent kit of ethanol determination liquid, which is a liquid double reagent formed by reagent I and reagent II, wherein the reagent I includes buffer solution (pH8.5-9.5)0.05-0.5M, reaction promoter 1-200g/L, stabilizer 0.1-10g/L, and preservative 0.1-10g/L; and reagent II includes buffer solution (pH6.0-7.0)0.05-0.5M, beta-coenzymeI 0.1-10g/L, alcohol dehydrogenase 0.2-200KU/L, compound stabilizer 0.1-10g/L, activator 0.1g-100g/L, and preservative 0.1-10g/L. The invention has the advantages that the reagent kit is highly accurate, precise and stable, can be stably stored at least for 12 months away from the light, and can fully satisfy the requirements of clinic detection.
Description
Technical field
The invention belongs to medical test determination techniques field, be specifically related to a kind of stable alcohol determining liquid reagent box.
Background technology
Ethanol (alcohol) is present in most of alcoholic drink, and the speed of its metabolism depends primarily on the content of body endoenzyme, has bigger individual difference.As everyone knows, low alcohol consumption can stimulate circulation, but a large amount of alcohol absorption then can cause alcohol dependence, can cause the infringement of spirit and multiple organ dysfunction, and is wherein to see the harm of liver function and moral function more, also serious.The habituation property of ethanol, abuse and dependence are one of serious social economic and public health problems in world today's scope, and in western countries, alcohol correlativity hepatopathy has become dead major reason.Therefore the blood alcohol determining plays a part very important in acute alcoholism diagnosis and medical jurisprudence evaluation.
The method for quantitatively determining of ethanol mainly contains chemical colorimetry, enzyme terminal colorimetric analysis, homogeneous EIA method, vapor-phase chromatography and expiratory air ethanol analytic approach etc. in the biological sample.Vapor-phase chromatography is considered to the reference method of alcohol determining.Vapor-phase chromatography at first with the NaCl saturated solution in the container of a sealing with serum or diluting blood sample, be the gas of volatilization on the top of closed container.Sampling makes sample cleaning like this, and is very little or do not have a matrix influence.Can accomplish the quantitative of peak value through typical curve.Shortcoming is that instrument is expensive, and the complicated inconvenience of formality is promoted.The enzyme process autoanalyzer method divides oxidation of ethanol enzyme process and alcohol dehydrogenase enzyme process, and it is simple that oxidation enzyme process reagent set becomes, and sample does not need pre-service, but poor specificity, methyl alcohol disturbs greatly measuring, and false positive can occur.Alcohol dehydrogenase is special relatively for ethanol; Can not play catalytic reaction with methyl alcohol, acetone or isopropyl alcohol etc.; Therefore accuracy is higher with precision, and it can be at the ultraviolet analyser or partly, measure on the automatic clinical chemistry analyzer, speed is fast; Good with the vapor-phase chromatography correlativity, its principle is following:
Ethanol+DPN
+ 
Acetaldehyde+DPNH+H
+
Generate DPNH with DPN when being generated acetaldehyde by the alcohol dehydrogenase oxydasis according to ethanol; The DPNH that produces can be directly in 340 nm place colorimetric detection; Utilize the enzyme process of DPNH similar with many other enzyme process just in use; Can on the existing instrument in laboratory, accomplish, this is medically having suitable sensitivity.
Enzyme assay has the specificity height; But advantage simple and quick, accurate safe automated analysis; But because alcohol dehydrogenase is extremely unstable; But high concentration stable existence several days only 5 ℃ time the in nearly neutral pure water is unstable in acid (pH is lower than 6.0) and alkalescence (pH is greater than 8.5) solution, and heavy metal with at present generally the reaction promoter example hydrochloric acid semicarbazides of use, glycocoll etc. has obvious inhibiting effect to it; In addition, the DPN that uses in the reagent is unstable under this reaction conditions; Therefore must to process powdered reagent could long preservation stable for this reagent; Also must use as early as possible after the redissolution; Otherwise in the preservation process,, can't satisfy the Clinical detection needs because alcohol dehydrogenase and DPN degradation speed comparatively fast can make a big impact to the reagent performance.
Summary of the invention
The present invention is directed to the deficiency of prior art, provide a kind of accuracy, precision all better, good stability, 2～8 ℃ keep in Dark Place and can stablize 12 months at least, can satisfy the stable alcohol determining liquid reagent box of clinical examination requirement fully.
In order to solve the problems of the technologies described above, the technical scheme that the present invention adopted is: stable alcohol determining liquid reagent box, this kit are the liquid-type double reagent of being made up of reagent 1 and reagent 2, wherein
Reagent 1 each component and concentration are:
Damping fluid (pH 8.5-9.5) 0.05 ~ 0.5 M (mol/L)
Reaction promoter 1 ~ 200 g/L
Stabilizing agent 0.1 ~ 10 g/L
Antiseptic 0.1 ~ 10 g/L;
Reagent 2 each component and concentration are:
Damping fluid (pH 6.0-7.0) 0. 05 ~ 0.5 M
β-DPN (claiming DPN or NADH again) 0.1 ~ 10 g/L
Alcohol dehydrogenase 0.2 ~ 200 KU/L
Compound stabilizer 0.1 ~ 10 g/L
Activator 0.1 ~ 100 g/L
Antiseptic 0.1 ~ 10 g/L.
Damping fluid in the mentioned reagent 1 of the present invention is Tri(Hydroxymethyl) Amino Methane Hydrochloride damping fluid, 3-Pehanorm-2-hydroxy-propanesulfonic acid damping fluid, 4-hydroxyethyl piperazine propane sulfonic acid damping fluid; A kind of in the sodium phosphate buffer etc., preferred 4-hydroxyethyl piperazine propane sulfonic acid damping fluid.
Reaction promoter in the mentioned reagent 1 of the present invention is one or more (i.e. two kinds or the two or more mixing, the proportion relation between several kinds of components of mixing does not require) in succinic acid, semicarbazide hydrochloride, glycocoll, two glycosides peptide, the acetaldehyde dehydrogenase etc.
Stabilizing agent in the mentioned reagent 1 of the present invention be polysorbas20, Tween 80, Bu Lijie-35 (
Brij-35), in Qu Latong 100 non-ionic surfactants such as (triton x-100s) one or more.
Damping fluid in the mentioned reagent 2 of the present invention is a kind of in hydroxyethyl piperazine second thiosulfonic acid damping fluid, 4-hydroxyethyl piperazine propane sulfonic acid damping fluid, Tri(Hydroxymethyl) Amino Methane Hydrochloride damping fluid, the 3-Pehanorm-2-hydroxy-propanesulfonic acid damping fluid etc.; Preferred hydroxyethyl piperazine second thiosulfonic acid damping fluid, pH 6.0-7.0.
Above-mentioned compound stabilizer is one or more and the enzyme combining stabilizer M03 (ES-W03) in glutathione, halfcystine, sucrose, trehalose or the seralbumin; Enzyme combining stabilizer M07 (ES-W07); A kind of composition among the enzyme combining stabilizer M08 (ES-W08) (be compound stabilizer be from glutathione, halfcystine, sucrose, trehalose or seralbumin one or more again with enzyme combining stabilizer M03; Enzyme combining stabilizer M07, a kind of among the enzyme combining stabilizer M08 are mixed together use).
Above-mentioned activator is one or more (i.e. two kinds or the two or more mixing, the proportion relation between several kinds of components of mixing does not require) in mercaptoethanol, DTT, halfcystine and the ethane diacid tetraacethyl disodium.
Antiseptic of the present invention is Sodium azide or proclin series antiseptic (like Proclin300) etc.
The compound method of reagent of the present invention 1 and reagent 2 is a conventional method, promptly according to above-mentioned formula rate, respectively reagent 1 and reagent 2 described each component is added respectively to mix to stir separately behind the distilled water and gets final product.
The test condition of the ethanol in the kit measurement sample of the present invention is following: temperature: 30-37 ℃; The cuvette optical path is 1.0 cm.Detect predominant wavelength 340 nm.
The method of using ethanol in the alcohol determining kit measurement sample of the present invention is following: sample (calibration tube is made sample with calibration object) adds reagent 1 mixing; 30-37 ℃ is read absorbance A 0 after hatching 3-5 min; Add reagent 2 mixings immediately; Behind 30-37 ℃ of reaction 5 min, read absorbance A 1, Δ A=A1-A0.Sample consumption 2 μ l wherein, reagent 1 consumption 200 μ l, reagent 2 consumptions 50 μ l.
Ethanol content calculates by following formula in the kit measurement sample of the present invention:
Outstanding substantive distinguishing features of the present invention and marked improvement mainly show:
(1) the present invention has protected alcohol dehydrogenase and the DPN stability in pH 6.0-7.0 damping fluid through in reagent 2, adding compound stabilizer, and reagent 2-8 ℃ preservation of the present invention can be stablized 12 months at least, can satisfy the clinical examination requirement fully.
(2) the present invention has evaded the inhibiting effect of reaction promoter to alcohol dehydrogenase to a certain extent through in reagent 2, adding activator.
?
Embodiment
Below be specific embodiment of the present invention, technical scheme of the present invention is further described, but the present invention is not limited to these embodiment.
Detection principle of the present invention is following: generate DPNH with DPN when being generated acetaldehyde according to ethanol by the alcohol dehydrogenase oxydasis; The DPNH that produces can be directly in 340 nm place colorimetric detection, and the absorbance rate of change is directly proportional with concentration of alcohol in the sample.
Embodiment 1
Reagent 1 each component and concentration are:
Sodium phosphate buffer (pH 8.5) 0.1 M
Semicarbazide hydrochloride 2 g/L
Polysorbas20 1 g/L
Proclin300 0.1?g/L；
Reagent 2 each component and concentration are:
Tri(Hydroxymethyl) Amino Methane Hydrochloride damping fluid (pH 6.0) 0. 05 M
Cozymase 0.5 g/L
Alcohol dehydrogenase 20 KU/L
Trehalose (compound stabilizer) 10 g/L
ES-W03 (compound stabilizer) 10 g/L
Mercaptoethanol (activator) 2 g/L
Proclin300 0.2?g/L。
The compound method of reagent 1 and reagent 2 is a conventional method, and promptly reagent 1 and reagent 2 said components add respectively to mix to stir separately behind the distilled water and get final product.
Embodiment 2
Reagent 1 each component and concentration are:
Tri(Hydroxymethyl) Amino Methane Hydrochloride damping fluid (pH 9.5) 0.05 M
Two glycosides peptide 5 g/L
Tween 80 10 g/L
Proclin300 0.2?g/L；
Reagent 2 each component and concentration are:
3-Pehanorm-2-hydroxy-propanesulfonic acid damping fluid (pH 6.5) 0.1 M
Cozymase 5 g/L
Alcohol dehydrogenase 50 KU/L
Halfcystine (compound stabilizer) 5 g/L
ES-W07 (compound stabilizer) 1 g/L
DTT (activator) 10 g/L
Sodium azide 0.1 g/L.
The preparation method of embodiment 2 kits is with embodiment 1.
Embodiment 3
Reagent 1 each component and concentration are:
4-hydroxyethyl piperazine propane sulfonic acid damping fluid (pH 9.5) 0.1 M
Glycocoll 10 g/L
Bu Lijie-35 5 g/L
Sodium azide 0.1 g/L
Reagent 2 each component and concentration are:
Hydroxyethyl piperazine second thiosulfonic acid damping fluid (pH 7.0) 0.1 M
Cozymase 5 g/L
Alcohol dehydrogenase 50 KU/L
Glutathione (compound stabilizer) 2 g/L
ES-W08 (compound stabilizer) 2 g/L
Halfcystine (activator) 10 g/L
Ethane diacid tetraacethyl disodium (activator) 1 g/L
Proclin300 0.1?g/L。
The preparation method of embodiment 3 kits is with embodiment 1.
Reference examples 1
Prepare by embodiment 3, but do not add compound stabilizer glutathione and ES-W08.
Reference examples 2
Prepare by embodiment 3, but do not add activator ethane diacid tetraacethyl disodium.
The raw material that embodiment is used unless otherwise indicated, is commercially available industrial goods.
The test condition of the ethanol in the kit measurement sample of the present invention is following: temperature: 30-37 ℃; The cuvette optical path is 1.0 cm.Detect predominant wavelength 340 nm.
The method of using ethanol in the alcohol determining kit measurement sample of the present invention is following: sample (calibration tube is made sample with calibration object) adds the R1 mixing; 30-37 ℃ is read absorbance A 0 after hatching 3-5 min, adds the R2 mixing immediately, behind 30-37 ℃ of reaction 5 min; Read absorbance A 1, Δ A=A1-A0.Sample consumption 2 μ l wherein, reagent 1 consumption 200 μ l, reagent 2 consumptions 50 μ l.
Ethanol content calculates by following formula in the kit measurement sample of the present invention:
Describe below in conjunction with the performance of form embodiment of the invention gained kit.
1, reaction rate
With embodiment 3, reference examples 1 detects sample with the reagent of reference examples 2 preparations, and it is as shown in table 1 that the sample absorbance changes (Δ A mensuration-Δ A is blank) testing result:
Table 1
| Group | Reference examples 1 | Reference examples 2 | Embodiment 3 |
| Δ A mensuration-Δ A is blank | 1.6123 | 1.0368 | 1.6261 |
Above data show that obviously reference examples 1 of the present invention is very fast with the reagent reacting speed of embodiment 3 preparations, add activator and have evaded the inhibiting effect of reaction promoter to alcohol dehydrogenase to a certain extent.
2, kit stability experiment
(1) 37 ℃ of accelerated stability experiment
The reagent of reference examples 1-2 and embodiment 3 preparations is carried out 37 ℃ of accelerated stabilities experiments (but 37 ℃ of accelerated stability tests 7 days simulating reality 4-8 ℃ preserved 1 year), and it is as shown in table 2 that the sample absorbance changes (Δ A mensurations-Δ A blank) testing result:
Table 2
| Time | Reference examples 1 | Reference examples 2 | Embodiment 3 |
| The 1st day | 1.6431 | 1.1322 | 1.6481 |
| 37 ℃ 3 days | 1.0214 | 1.1164 | 1.5946 |
| 37 ℃ 5 days | 0.6532 | 0.9845 | 1.4987 |
| 37 ℃ 7 days | 0.2847 | 0.9063 | 1.4652 |
| The 7th day decline % | 82.7％ | 20.0% | 11.1% |
(2) 4-8 ℃ of stability experiment
The reagent of reference examples 1-2 and embodiment 3 preparations was preserved 1 year at 4-8 ℃, and it is as shown in table 3 that the sample absorbance changes (Δ A mensuration-Δ A is blank) result:
Table 3
| Month | Reference examples 1 | Reference examples 2 | Embodiment 3 |
| 1 | 1.6324 | 1.2104 | 1.6358 |
| 3 | 1.2546 | 1.1865 | 1.6124 |
| 5 | 0.9894 | 1.1654 | 1.5864 |
| 7 | 0.7605 | 1.1320 | 1.5623 |
| 9 | 0.5231 | 1.1006 | 1.5417 |
| 12 | 0.3746 | 1.0836 | 1.5103 |
Above data declaration, reference examples 2 of the present invention is better with the reagent stability of embodiment 3 preparations, and the stability of alcohol dehydrogenase and DPN has been protected in the adding of compound stabilizer.
Claims (10)
1. stable alcohol determining liquid reagent box is characterized in that: the liquid-type double reagent of this kit for forming by reagent 1 and reagent 2, wherein
Reagent 1 each component and concentration are:
Damping fluid (pH 8.5-9.5) 0.05 ~ 0.5 M
Reaction promoter 1 ~ 200 g/L
Stabilizing agent 0.1 ~ 10 g/L
Antiseptic 0.1 ~ 10 g/L;
Reagent 2 each component and concentration are:
Damping fluid (pH 6.0-7.0) 0. 05 ~ 0.5 M
β-DPN 0.1 ~ 10 g/L
Alcohol dehydrogenase 0.2 ~ 200 KU/L
Compound stabilizer 0.1 ~ 10 g/L
Activator 0.1 g ~ 100 g/L
Antiseptic 0.1 ~ 10 g/L.
2. stable alcohol determining liquid reagent box according to claim 1 is characterized in that: the damping fluid in the reagent 1 is a kind of in Tri(Hydroxymethyl) Amino Methane Hydrochloride damping fluid, 3-Pehanorm-2-hydroxy-propanesulfonic acid damping fluid, 4-hydroxyethyl piperazine propane sulfonic acid damping fluid, the sodium phosphate buffer.
3. stable alcohol determining liquid reagent box according to claim 2 is characterized in that: the damping fluid in the reagent 1 is a 4-hydroxyethyl piperazine propane sulfonic acid damping fluid.
4. stable alcohol determining liquid reagent box according to claim 1 is characterized in that: the reaction promoter in the reagent 1 is one or more in succinic acid, semicarbazide hydrochloride, glycocoll, two glycosides peptide, the acetaldehyde dehydrogenase.
5. stable alcohol determining liquid reagent box according to claim 1 is characterized in that: the stabilizing agent in the reagent 1 is one or more among polysorbas20, Tween 80, Bu Lijie-35, the Qu Latong 100.
6. stable alcohol determining liquid reagent box according to claim 1 is characterized in that: the damping fluid in the reagent 2 is a kind of in hydroxyethyl piperazine second thiosulfonic acid damping fluid, 4-hydroxyethyl piperazine propane sulfonic acid damping fluid, Tri(Hydroxymethyl) Amino Methane Hydrochloride damping fluid, the 3-Pehanorm-2-hydroxy-propanesulfonic acid damping fluid.
7. stable alcohol determining liquid reagent box according to claim 6 is characterized in that: the damping fluid in the reagent 2 is a hydroxyethyl piperazine second thiosulfonic acid damping fluid.
8. stable alcohol determining liquid reagent box according to claim 1; It is characterized in that: the compound stabilizer described in the reagent 2 is one or more and the enzyme combining stabilizer M03 in glutathione, halfcystine, sucrose, trehalose or the seralbumin; Enzyme combining stabilizer M07, a kind of mixing among the enzyme combining stabilizer M08 is used.
9. stable alcohol determining liquid reagent box according to claim 1 is characterized in that: described activator described in the reagent 2 is one or more in mercaptoethanol, DTT, halfcystine, the ethane diacid tetraacethyl disodium.
10. stable alcohol determining liquid reagent box according to claim 1 is characterized in that: described antiseptic is Sodium azide or proclin series antiseptic 2.
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|---|---|---|---|
| CN 201110347261 CN102495220B (en) | 2011-11-07 | 2011-11-07 | Stable reagent kit of ethanol determination liquid |
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|---|---|---|---|
| CN 201110347261 CN102495220B (en) | 2011-11-07 | 2011-11-07 | Stable reagent kit of ethanol determination liquid |
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| CN102495220B CN102495220B (en) | 2013-11-06 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106885803A (en) * | 2015-12-16 | 2017-06-23 | 山东博科生物产业有限公司 | A kind of strong antijamming capability and degree of accuracy enzyme process ethanol detection reagent high |
| CN111948384A (en) * | 2019-05-23 | 2020-11-17 | 裴广华 | Sucrose-containing biological product stabilizer for ELISA kit |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106885803A (en) * | 2015-12-16 | 2017-06-23 | 山东博科生物产业有限公司 | A kind of strong antijamming capability and degree of accuracy enzyme process ethanol detection reagent high |
| CN106885803B (en) * | 2015-12-16 | 2019-06-28 | 山东博科生物产业有限公司 | A kind of strong antijamming capability and the high enzyme process ethyl alcohol detection reagent of accuracy |
| CN111948384A (en) * | 2019-05-23 | 2020-11-17 | 裴广华 | Sucrose-containing biological product stabilizer for ELISA kit |
| CN111948384B (en) * | 2019-05-23 | 2024-02-27 | 裴广华 | Sucrose-containing biologicals stabilizer for ELISA kit |
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Address after: 315104 Zhejiang city of Ningbo province Yinzhou District Qiming Road No. 299 Patentee after: MEDICAL SYSTEM BIOTECHNOLOGY Co.,Ltd. Address before: 315104 Ningbo, Yinzhou, Central District, No. Qiming South Road, No. 299 Patentee before: NINGBO MEDICAL SYSTEM BIOTECHNOLOGY Co.,Ltd. |
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Application publication date: 20120613 Assignee: Ningbo Shengyuan Biotechnology Co., Ltd. Assignor: MEDICAL SYSTEM BIOTECHNOLOGY Co.,Ltd. Contract record no.: X2022330000361 Denomination of invention: Stable ethanol assay liquid kit Granted publication date: 20131106 License type: Common License Record date: 20220809 |