CN101504373A - Liquid stabilized serum cholinesterase reagent - Google Patents
Liquid stabilized serum cholinesterase reagent Download PDFInfo
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- CN101504373A CN101504373A CNA2009101138893A CN200910113889A CN101504373A CN 101504373 A CN101504373 A CN 101504373A CN A2009101138893 A CNA2009101138893 A CN A2009101138893A CN 200910113889 A CN200910113889 A CN 200910113889A CN 101504373 A CN101504373 A CN 101504373A
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- cholinesterase
- serum cholinesterase
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Abstract
The invention discloses a serum cholinesterase reagent with low toxicity, good stability, accurate detection result, easy storage and steady liquid, which consists of a reagent 1 and a reagent 2, wherein the function of the reagent 1 is to adjust the pH value of the reaction of a working reagent, the reagent 2 is stabilized cholinesterase substrate solution, and the reagent 1 and the reagent 2 form an integral detection reagent during the detection. Compared with the prior art, the serum cholinesterase reagent takes an epoxy ethane condensation compound and sulfate as a stabilizing agent of the cholinesterase substrate solution, so that the serum cholinesterase reagent has good stability performance, the storage life of the reagent can reach more than 15 months at a temperature of between 4 and 8 DEG C, and the serum cholinesterase reagent does not conflict with the prior reagent bottle which is made of organic materials and is matched with a full-automatic biochemical analyzer; the reagent has low toxicity, so the reagent almost has no pollution during the use and scrap disposal, and the use risk of operators is greatly reduced; and at the same time, the reagent also has the advantages of simple and convenient and quick operation, easy storage and the like.
Description
Technical field:
The present invention relates to the mensuration reagent of cholinesterase in the serum, be specially a kind of serum cholinesterase reagent of liquid stabilising.
Background technology:
CHE is the adopted name of enzymatic activity, and according to the difference of effect, cholinesterase also can be subdivided into acetylcholine esterase and acyl group CHE.Acyl group CHE (CHE) mainly is present in pancreas, heart, liver, the serum.Clinic study is found; acyl group activity of cholinesterase among the patient of organophosphorus insecticide poisoning and chronic liver disease in the body has obvious decline, there are some researches show that 4/5ths serious hepatitis patient CHE reduces to normal 60%; it is normal 10% that the urgent patient can reduce to, even disappearance fully.In addition, chronic active hepatitis etc. all can cause the CHE vigor to descend, and measure therefore that CHE estimates liver function and the prediction of hepatopathy has important value.
The most general in the acyl group CHE substrate with bytyry thiocholine (BTC) or propiono thiocholine, wherein BTC is considered to CHE one of specific substrate the most, so mensuration for the cholinesterase in human serum and the body fluid, laboratory medicine circle mainly adopts BTC to make substrate, the thiocholine that discharges after the enzymatic reaction and 5,5 '-two sulphur two (2-nitrobenzoic acids) (DTNB) reacts the compound 5-sulfo--2-nitrobenzoic acid (absorption maximum is at 410nm) that generates yellow, adopt rate method or end-point method to measure (Gary, P.G., Cholinesterase determination.CLIN.CHEM.1971; 17,1145～1148).Yet, because above-mentioned CHE substrate stability in aqueous solution is very low, in aqueous solution,, also very easily be hydrolyzed into organic acid and thiocholine even without cholinesterase catalysis, thus the accuracy of disturbing reagent to detect.Therefore, how making CHE substrate solution stabilization is to keep CHE to measure reagent stability, guarantee the true and reliable key of measurement result.
At this type of CHE substrate solution stabilization problem, State Intellectual Property Office's disclosed publication number on August 2nd, 2006 is CN1811393A, name is called the patent of invention of " serum cholinesterase reagent of liquid stabilising ", disclose with 0.1mmol/L to the phenol solution of saturation concentration as the BTC stabilization technique of solution and relevant cholinesterase liquid detectable.But there is following deficiency in this method: phenol solubleness in water is low, and easy temperature influence, causes precipitation to disengage in the low-temperature storage process easily; And phenol is exposed to and is oxidized to aldehyde ketone in the air easily, causes in solution changes color; Thereby cause such reagent in use to be prone to phenomenons such as the fluctuation of reagent blank instability, measurement result is big, strengthened the uncertainty of testing result.In addition, publication number is CN1978660A, the patent of invention that name is called " stabilized cholinesterase substrate solution " discloses a kind of polar organic solvent that utilizes as the BTC stabilization technique of stable and relevant cholinesterase liquid detectable, and specifically having set forth certain density polar organic solvent such as ethanol, acetone, dimethyl sulfoxide (DMSO), acetone, diacetone, tetrahydrofuran etc. has good stabilization to BTC.Though this method has improved the stability of BTC solution to a great extent, but because this method has been used the polar organic solvent of big consumption, and there are shortcomings such as the big or bio-toxicity of volatility is strong in such organic solvent, therefore this class methods reagent all can cause in various degree harm to operator's health in use, and reagent also easily causes environmental pollution scrapping when handling.The polar organic solvent solution of adopting higher concentration simultaneously is not suitable for depositing in organic material container (as polyethylene kind plastic bottle etc.) and using, and this all adopts the actual conditions of organic material to contradict with present most automatic clinical chemistry analyzer matched reagent bottles.
Summary of the invention:
The technical problem to be solved in the present invention is: at the deficiency that CHE reagent in the prior art is stable inadequately, toxicity is bigger, provide a kind of low toxicity, good stability, testing result accurately and the serum cholinesterase reagent of the liquid stabilising that is easy to deposit.
Technical scheme of the present invention: a kind of serum cholinesterase reagent of liquid stabilising, form by reagent one and reagent two, wherein, reagent one comprises:
The pH value is buffer solution 20～200mmol/L of 6.5～7.8;
Sulfydryl developer 0.5～5mmol/L;
Described reagent two comprises:
The pH value is acidic buffer 20～200mmol/L of 3.5～5.0;
Ethylene oxide condensate 5～100g/L;
Sulfate 5～100g/L;
Cholinesterase substrate 10～50g/L.
Among the present invention, the effect of reagent one is the ph value of reaction of adjusting work reagent, and second reagent be the cholinesterase substrate solution of stabilization, and both form complete detectable when detecting.Reagent one with the proportionate relationship of reagent two is: reagent two mixes formation work reagent with reagent one, and to make the pH value of this work reagent be 7.4～7.8, and generally the blending ratio of the two (volume) is a reagent one: reagent two=30:1.
In order to improve sample serum, the blood plasma dissolubility in reagent and the permeability of detection reaction system, can in reagent one and/or reagent two, add a certain amount of non-ionics respectively, its consumption is generally at 0.5～1g/L; Specifically can be Qu Latong X-100, tween-20, tween-80 or the bay ether 35 etc. of concentration in this scope; Simultaneously in order to prevent reagent long bacterium in storage period, also can in reagent one and/or reagent two, add a spot of antiseptic respectively, as antiseptics commonly used such as acetamide chlcride, ethyl acetate sodium or ethyl benzoate sodium, the consumption of described antiseptic is controlled at 0.1～0.5g/L.
In the technique scheme, wherein in the reagent one:
Described buffer solution can be pH value phosphate buffer, borate buffer solution or GOOD ' S biological buffer in 6.5～7.8 scopes; Wherein, GOOD ' S biological buffer can be PIPES, MOPSO, MOPS, BES or HEPES damping fluid etc.;
Described sulfydryl developer can be 5,5-two thiobiss (2-nitrobenzoic acid), dihexyl nabam or phenanthroline.
In reagent two:
Described acidic buffer can be at least a in phosphate buffer, acetate buffer solution and GOOD ' the S biological buffer of pH value in 3.5～5.0 scopes; Wherein, GOOD ' S biological buffer can be MOPSO, MOPS or HEPES damping fluid etc.;
Described ethylene oxide condensate is that chemical general formula is HO (CH
2CH
2O) nH, the molecular weight ethylene glycol polyoxyethylene ether (PEG) between 190～8500Dalton; Specifically can be selected from any one or two or more combinations among PEG200, PEG400, PEG600, PEG800, PEG1000, PEG1500, PEG2000, PEG3000, PEG4000, PEG6000 and the PEG8000;
Described sulfate is to be selected from least a in sodium sulphate, glazier's salt, niter cake, potassium acid sulfate and the sulfate of ammoniac;
Described cholinesterase substrate is bytyry thiocholine or propiono thiocholine.
Reagent detection reaction principle of the present invention is as follows:
Calculate the enzyme activity of cholinesterase in the sample by the absorbance ascending velocity of detection under the 410nm wavelength.
From mentioned reagent box detection reaction principle as can be known one of cholinesterase substrate hydrolysate thiocholine can with 5-sulfo--2-nitrobenzoic acid reaction solution.Therefore can be by detecting different times and different holding conditions reagent two (cholinesterase substrate solution) down and reagent one mixed absorbance variation, promptly the reagent blank absorbance changes the stability of verifying cholinesterase substrate solution.
The applicant is placing storage period in the stability experiment of preserving 15 months under 15 days stable load test and 4 to the 8 ℃ of storage temperatures with reagent one and reagent two at 37 ℃, reagent blank absorbance and reagent detect the factor and have no significant change, reagent all has no effect the clinical detection of loading fully requirement on performance index such as accuracy, precision, the range of linearity.
Reagent of the present invention can adopt conventional manual assay method, also can detect sample by analysis instruments such as automatic clinical chemistry analyzers, specifically can adopt following method:
Method 1:
Mixing, 37 ℃ of 60 seconds time delays, minute 120 seconds is measured △ A/ branch.
Computing method: CHE (U/L)=△ A/ branch * F
Wherein F is the calculated factor that reagent is measured, and in order to guarantee reliability of testing result, need before pattern detection to require by CHE standard items or standard serum the F factor to be carried out the school card.
Method 2:
Before mensuration with reagent one and reagent two by volume for the ratio of 30:1 is mixed into single reagent, detect by the following method, computing method are the same.
Mixing, 37 ℃ of 60 seconds time delays, minute 120 seconds is measured △ A/ branch at 405nm.
Sample described in the said determination method can be serum or blood plasma.
Compared with prior art, the present invention is with ethylene oxide condensate and the sulfate stabilizing agent as cholinesterase substrate solution, not only make the stability of serum cholinesterase reagent good, reagent can reach more than 15 months 4～8 ℃ of temperature following storage lives, and is not inconsistent with the automatic clinical chemistry analyzer matched reagent bottle that existing employing organic material is made; Therefore and, using and scrapping almost pollution-free in the process of processing, operator's application risk and reduce greatly environment because reagent toxicity is low; Simultaneously reagent of the present invention also has easy and simple to handle, quick, advantage such as be easy to deposit.
Embodiment:
The invention will be further described with embodiment below, but the present invention is not limited to these embodiment.
Embodiment 1
Reagent one:
Reagent component concentration
The potassium dihydrogen phosphate of pH=7.8-sodium hydrogen phosphate damping fluid 100mmol/L
5,5-two thiobiss (2-nitrobenzoic acid) 1mmol/L
Qu Latong X-100 0.5g/L
Acetamide chlcride 0.2g/L
Reagent two:
Reagent component concentration
The HEPES damping fluid 50mmol/L of pH=4.5
PEG200 20g/L
PEG6000 10g/L
Sodium sulphate 5g/L
Niter cake 10g/L
Butyryl thiocholine 16g/L
Reagent one and reagent two 37 ℃ place 15 days after, concentration of substrate, sulfydryl developer and reagent performance do not have obviously and fall, when detecting according to assay method of the present invention, the calculated factor of reagent is between 17000～18000, and the enzyme activity determination upper limit can reach 12000～13000U/L.
Embodiment 2
Reagent one:
Reagent component concentration
The MOPOS damping fluid 50mmol/L of pH=7.5
Dihexyl nabam 1mmol/L
Bay ether 35 1g/L
Acetamide chlcride 0.2g/L
Reagent two:
Reagent component concentration
The potassium phosphate buffer 200mmol/L of pH=4.5
PEG400 20g/L
PEG6000 50g/L
Sulfate of ammoniac 80g/L
Niter cake 20g/L
Butyryl thiocholine 10g/L
Reagent one and reagent two 37 ℃ place 15 days after, concentration of substrate, sulfydryl developer and reagent performance do not have obviously and fall, when detecting according to assay method disclosed by the invention, the calculated factor of reagent is between 15000～17000, and the enzyme activity determination upper limit can reach 12000～13000U/L.
Embodiment 3
Reagent one:
Reagent component concentration
The potassium dihydrogen phosphate of pH=6.5-sodium hydrogen phosphate damping fluid 50mmol/L
5,5-two thiobiss (2-nitrobenzoic acid) 3mmol/L
Reagent two:
Reagent component concentration
The potassium phosphate buffer 100mmol/L of pH=5.0
PEG6000 5g/L
Sodium sulphate 10g/L
Potassium acid sulfate 20g/L
Propionylthiocholine 25g/L
Reagent one and reagent two 37 ℃ place 15 days after, concentration of substrate, sulfydryl developer and reagent performance do not have obviously and fall, when detecting according to assay method disclosed by the invention, the calculated factor of reagent is between 21000～22000, and the enzyme activity determination upper limit can reach 11000～12000U/L.
Embodiment 4
Reagent one:
Reagent component concentration
PH=7.5 potassium dihydrogen phosphate-sodium hydrogen phosphate damping fluid 50mmol/L
5,5-two thiobiss (2-nitrobenzoic acid) 5mmol/L
Tween-80 1g/L
Reagent two:
Reagent component concentration
PH=3.5 acetic acid-sodium-acetate buffer 100mmol/L
PEG6000 30g/L
PEG8000 10g/L
Sodium sulphate 20g/L
Potassium acid sulfate 15g/L
Butyryl thiocholine 50g/L
Reagent one and reagent two 37 ℃ place 15 days after, concentration of substrate, sulfydryl developer and reagent performance do not have obviously and fall, when detecting according to assay method disclosed by the invention, the calculated factor of reagent is between 15000～18000, and the enzyme activity determination upper limit can reach 12000～13000U/L.
Embodiment 5
Reagent one:
Reagent component concentration
The potassium dihydrogen phosphate of pH=7.5-sodium hydrogen phosphate damping fluid 50mmol/L
Phenanthroline 1mmol/L
Bay ether 35 1g/L
Ethyl acetate sodium 0.2g/L
Reagent two:
Reagent component concentration
The acetic acid of pH=4.0-sodium-acetate buffer 100mmol/L
PEG6000 30g/L
PEG8000 70g/L
Glazier's salt 20g/L
Niter cake 40g/L
Butyryl thiocholine 30g/L
Tween-20 0.8g/L
Methyl phenyl ethers anisole ethyl ester sodium 1g/L
Reagent one and reagent two 37 ℃ place 15 days after, concentration of substrate, sulfydryl developer and reagent performance do not have obviously and fall, when detecting according to assay method disclosed by the invention, the calculated factor of reagent is between 15000～18000, and the enzyme activity determination upper limit can reach 12000～13000U/L.
Embodiment 6
Reagent one:
Reagent component concentration
The MOPOS damping fluid 50mmol/L of pH=7.8
Dihexyl nabam 0.5mmol/L
Tween-80 0.5g/L
Ethyl benzoate sodium 1g/L
Reagent two:
Reagent component concentration
PH=5.0 phosphate buffer 25mmol/L
PH=5.0MOPS damping fluid liquid 25mmol/L
PEG200 10g/L
PEG6000 25g/L
Sodium sulphate 20g/L
Potassium acid sulfate 15g/L
Butyryl thiocholine 16g/L
Reagent one and reagent two 37 ℃ place 15 days after, concentration of substrate, sulfydryl developer and reagent performance do not have obviously and fall, when detecting according to assay method disclosed by the invention, the calculated factor of reagent is between 18000～20000, and the enzyme activity determination upper limit can reach 12000～13000U/L.
Embodiment 7
Reagent one:
Reagent component concentration
The HEPES damping fluid 50mmol/L of pH=7.0
5,5-two thiobiss (2-nitrobenzoic acid) 2.5mmol/L
Qu Latong X-100 0.5g/L
Reagent two:
Reagent component concentration
The HEPES damping fluid 25mmol/L of pH=4.0
PEG200 20g/L
PEG6000 10g/L
Sodium sulphate 5g/L
Propionylthiocholine 30g/L
Qu Latong X-100 0.5g/L
Reagent one and reagent two 37 ℃ place 15 days after, concentration of substrate, sulfydryl developer and reagent performance do not have obviously and fall, when detecting according to assay method disclosed by the invention, the calculated factor of reagent is between 19000～21000, and the enzyme activity determination upper limit can reach 11000～12000U/L.
Claims (8)
1, a kind of serum cholinesterase reagent of liquid stabilising is made up of reagent one and reagent two, it is characterized in that:
Described reagent one comprises:
The pH value is buffer solution 20～200mmol/L of 6.5～7.8;
Sulfydryl developer 0.5～5mmol/L;
Described reagent two comprises:
The pH value is acidic buffer 20～200mmol/L of 3.5～5.0;
Ethylene oxide condensate 5～100g/L;
Sulfate 5～100g/L;
Cholinesterase substrate 10～50g/L.
2, the serum cholinesterase reagent of liquid stabilising according to claim 1 is characterized in that: the buffer solution in the described reagent one is phosphate buffer, borate buffer solution or GOOD ' S biological buffer.
3, the serum cholinesterase reagent of liquid stabilising according to claim 1 is characterized in that: the sulfydryl developer in the described reagent one is 5,5-two thiobiss (2-nitrobenzoic acid), dihexyl nabam or phenanthroline.
4, the serum cholinesterase reagent of liquid stabilising according to claim 1 is characterized in that: the acidic buffer in the described reagent two is at least a in phosphate buffer, acetate buffer solution and GOOD ' the S biological buffer.
5, the serum cholinesterase reagent of liquid stabilising according to claim 1 is characterized in that: the ethylene oxide condensate in the described reagent two is that chemical general formula is HO (CH
2CH
2O) nH, the molecular weight ethylene glycol polyoxyethylene ether (PEG) between 190～8500 Dalton.
6, the serum cholinesterase reagent of liquid stabilising according to claim 5 is characterized in that: described ethylene oxide condensate is to be selected from least a among PEG200, PEG400, PEG600, PEG800, PEG1000, PEG1500, PEG2000, PEG3000, PEG4000, PEG6000 and the PEG8000.
7, the serum cholinesterase reagent of liquid stabilising according to claim 1 is characterized in that: the sulfate in the described reagent two is to be selected from least a in sodium sulphate, glazier's salt, niter cake, potassium acid sulfate and the sulfate of ammoniac.
8, the serum cholinesterase reagent of liquid stabilising according to claim 1 is characterized in that: the cholinesterase substrate in the described reagent two is bytyry thiocholine or propiono thiocholine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101138893A CN101504373B (en) | 2009-02-23 | 2009-02-23 | Liquid stabilized serum cholinesterase reagent |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101138893A CN101504373B (en) | 2009-02-23 | 2009-02-23 | Liquid stabilized serum cholinesterase reagent |
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| CN101504373A true CN101504373A (en) | 2009-08-12 |
| CN101504373B CN101504373B (en) | 2010-12-08 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102621138A (en) * | 2012-04-06 | 2012-08-01 | 上海蓝怡科技有限公司 | Preparation method of micro-emulsion kit |
| CN106018308A (en) * | 2016-08-11 | 2016-10-12 | 山东博科生物产业有限公司 | Cholinesterase detection kit |
-
2009
- 2009-02-23 CN CN2009101138893A patent/CN101504373B/en active Active
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102621138A (en) * | 2012-04-06 | 2012-08-01 | 上海蓝怡科技有限公司 | Preparation method of micro-emulsion kit |
| CN106018308A (en) * | 2016-08-11 | 2016-10-12 | 山东博科生物产业有限公司 | Cholinesterase detection kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101504373B (en) | 2010-12-08 |
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