CN105603049A - Compound stabilizer and kit for in vitro diagnosis reagents - Google Patents
Compound stabilizer and kit for in vitro diagnosis reagents Download PDFInfo
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- CN105603049A CN105603049A CN201610032329.5A CN201610032329A CN105603049A CN 105603049 A CN105603049 A CN 105603049A CN 201610032329 A CN201610032329 A CN 201610032329A CN 105603049 A CN105603049 A CN 105603049A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/58—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving urea or urease
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/50—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving creatine phosphokinase
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/52—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving transaminase
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a compound stabilizer and a kit for in vitro diagnosis reagents. The compound stabilizer comprises a sealing agent, a surfactant, a micromolecule organic reductant, a nucleic acid ingredient stabilizer, a biological active substance protecting agent and a biological preservative. The compound stabilizer can be universally applied to various in vitro diagnosis reagents and has better reagent stabilizing effect, and validity period of the reagents is prolonged effectively.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of answering for external diagnosis reagentClose stabilizing agent and kit.
Background technology
External diagnosis reagent is can be used alone or make with instrument, utensil, equipment or system in combinationWith, human sample is checked in vitro, detect for certain composition to sampleOr the material such as chemistry, biology or immunology of the in-vitro diagnosis check problem of measuring. External examiningDisconnected reagent system comprises detection reagent, reagent articles for use, caliberator (product), quality control substance (product)Etc. all multicomponents. External diagnosis reagent has been widely used in medical research and clinical examination,It is important step indispensable in conventional medical procedure. The reliable in-vitro diagnosis examination of steady qualityAgent can provide clinical examination data accurately, the prevention of disease and control and disease andTime diagnosis and treatment aspect brought into play important function.
The kind of external diagnosis reagent and of a great variety, can be divided into blood from clinical speciality classification, biochemistry, immunology, microbiology, cytohistology and molecular biology etc.; FromIn proterties, can be divided into liquid, dry powder, freeze-drying culture medium, detector bar etc.; Have with methodology classificationEnzyme exempts from, put exempt from, SABC, immunocytochemistry, fluorescence and chemiluminescence etc.; From detectingIn performance, can be divided into qualitative, sxemiquantitative, quantitative; Detection method, can be divided into chemical staining methodReagent, immunoturbidimetry reagent, enzyme process reagent, ELISA method reagent, colloidal gold method reagent,Cultivate base class reagent, supporting reagent, the CLIA reagent and poly-used of flow type analyzerSynthase chain reaction (PCR) reagent etc.
For the external diagnosis reagent of numerous kinds or kind, its production technology is widely different, rightThe quality requirement of product also emphasizes particularly on different fields; Meanwhile, because production model and the research and development of reagent are rawProduct personnel's technical capability difference, is easy to cause the reagent product quality of each producer irregularTogether. Wherein, very important quality problems are exactly the stability of product.
Due to the wide-scale distribution of the universal and technology of knowledge, those of ordinary skill can at this stageTo produce know-why and the method for certain diagnostic reagent and to make product than being easier to find. NewlyThe diagnostic reagent of preparation generally can reach basic fundamental requirement. But how to improve the stable of reagentProperty usually becomes the difficult point of research and development. Refer to as the prerequisite important technology of external diagnosis reagentMark, stability is to guarantee in use important indicator safely and effectively of reagent product. StableJournal of Sex Research need be according to the physicochemical property of reagent product, stabilizer element reasonable in design and content,To guarantee that the main quality index of reagent product continues to keep stable, Jin Erti over timeThe term of validity of high reagent product. Therefore, the selection of stabilizing agent and adding in external diagnosis reagent productAdding is a very complicated knowledge.
Chinese patent application (publication number: CN104946616A, title: a kind of for externalGeneral Pickering agent and the using method thereof of diagnostic reagent) one is disclosed for in-vitro diagnosisThe general Pickering agent of reagent. It obtains after by aqueous solution freeze-drying, described aqueous solution of raw materialComprise bovine serum albumin(BSA), EDETATE SODIUM salt or EDTA sylvite, sucrose or glycine or sweet dewSugar, polysorbas20 or polysorbate40 or Tween 80 or polyvinylpyrrolidone, cyclodextrin andPEG2000 or PEG6000 or PEG8000. The stabilizing agent that this invention provides should be to increasingThe stability of external diagnosis reagent plays a good role, but also has some other to carryThe material of high stability, this invention does not relate to. And described stabilizing agent is freeze-dried powder, itsNeed in use preparation, use and inconvenience. Chinese patent (publication number:CN104195221A, title: a kind of compound stabilizer for glucose assays reagent) public affairsOpened a kind of compound stabilizer for glucose assays reagent, it has stablized glucose assays examinationThe accuracy of agent testing result. But the stabilizing agent range of application that this invention provides is narrow, onlyThe stability of having improved glucose assays reagent. For problems of the prior art, haveThe new stabilizing agent of necessary exploitation further enriches the stability means of external diagnosis reagent.
Summary of the invention
The object of the present invention is to provide a kind of compound stabilizer for external diagnosis reagent, useThe not high defect of external diagnosis reagent stability in solving prior art.
Another object of the present invention be to provide a kind of comprise above-mentioned for external diagnosis reagentThe kit of compound stabilizer.
To achieve these goals, the invention provides a kind of compound for external diagnosis reagentStabilizing agent, it comprises sealer, surfactant, little molecule organic reducing agent, nucleic acid compositionsStabilizing agent, bioactivator protective agent and biological preservative; The content of described sealer is 1～55% (m/v), the content of described surfactant is 10.5～65% (v/v), described organicThe content of little molecule reducing agent is 0.02～1% (m/v), the containing of described nucleic acid compositions stabilizing agentAmount is 0.3～4% (m/v), and the protectant content of described bioactivator is 10～50%(m/v), the content of described biological preservative is 0.5～5% (v/v), and surplus is ultra-pure water.
In compound stabilizer provided by the invention, described sealer is preferably bovine serum albuminOne or more in casein, gelatin and skimmed milk power in vain. More preferably, described sealingAgent is bovine serum albumin(BSA) or/and casein, and the content of this sealer can be 7～18%(m/v). Most preferably, described sealer is bovine serum albumin(BSA) and casein, wherein, and instituteThe content of stating bovine serum albumin(BSA) can be 10% (m/v), and described caseic content canThink 1% (m/v).
In compound stabilizer provided by the invention, described surfactant is for reducing described multipleClose the surface tension of stabilizing agent. Preferably, described surfactant be polysorbas20, polysorbate60,One or more in Tween 80, propane diols, glycerine and polyethylene glycol PEG, Qi ZhongjuEthylene glycol PEG includes but not limited to PEG2000, PEG6000, PEG8000 etc. More excellentSelection of land, described surfactant is one in polysorbas20, polysorbate60, Tween 80 and propane diolsKind or multiple. Further preferably, described surfactant is polysorbas20 or/and propane diols,And the content of this surfactant is 11～52% (v/v). Most preferably, live in described surfaceProperty agent is polysorbas20 and propane diols, and wherein, the content of described polysorbas20 can be 2% (v/v),And the content of described propane diols can be 50% (v/v).
In compound stabilizer provided by the invention, described little molecule organic reducing agent is for albumenThe reduction of disulfide bond in matter, can be used for stoping the egg forming between the cysteine in proteinIn white matter molecule or intermolecular disulfide bond. Preferably, described organic molecule reducing agent comprise butBe not limited to thio-alcohol reducing agent. More preferably, described organic molecule reducing agent is two sulphur threosesOne or more in alcohol, TGA and 2 mercapto ethanol (being beta-mercaptoethanol), and shouldThe content of organic molecule reducing agent is 0.03～0.8% (m/v). Most preferably, described organicLittle molecule reducing agent is dithiothreitol (DTT), and its content can be 0.03% (m/v).
In compound stabilizer provided by the invention, described nucleic acid compositions stabilizing agent is for stable nucleusAcids composition. Preferably, described nucleic acid compositions stabilizing agent adopts borate, includes but not limited toMetaborate, ortho-borate, multi-borate etc. More preferably, described nucleic acid compositions stabilizing agentFor multi-borate, as sodium tetraborate, dipotassium tetraborate etc., and the containing of this nucleic acid compositions stabilizing agentAmount can be 0.76～3% (m/v). Most preferably, described nucleic acid compositions stabilizing agent is four boronAcid sodium, and its content can be 0.76% (m/v).
In compound stabilizer provided by the invention, described bioactivator protective agent is to biologyActive material plays a protective role. Preferably, described bioactivator protective agent adopts non-going backRaw sugar, includes but not limited to one or more in trehalose, sucrose, starch and cellulose,And its content can be 10～20% (m/v). More preferably, described biological active matter quality guaranteeProtecting agent is trehalose, and its content can be 20% (m/v).
In compound stabilizer provided by the invention, described biological preservative is preferably receives that he is mouldElement, polylysine, Sodium azide, thimerosal, ProClin150, ProClin300 and Proclin950In one or more, wherein the active component of ProClin series anticorrisive agent is mainly 2-methyl-4-isothiazoline-3-ketone (MCI) and CMIT (CMCI).More preferably, described biological preservative is ProClin150, ProClin300 and ProClin950In one or more, and its content can be 0.5～1% (v/v). Most preferably, instituteStating biological preservative is ProClin950, and its content can be 1% (v/v).
The preparation method of compound stabilizer provided by the invention is:
1) take respectively described sealer, little molecule organic reducing agent, nucleic acid by constituent contentStable components agent and bioactivator protective agent, fully dissolve above-mentioned each component, mix,Obtain solution 1;
2) measure respectively described surfactant and biological preservative by constituent content, and addIn solution 1, mix, obtain solution 2;
3) solution 2 use ultra-pure waters are carried out to constant volume, make described compound stabilizer.
On the other hand, the present invention also provides a kind of above-mentioned answering for external diagnosis reagent that compriseClose the kit of stabilizing agent.
Compared with existing technology, the beneficial effect of compound stabilizer provided by the invention is: provideMore materials that can improve stability, enriched the means that improve stability; Liquid is multipleClosing stabilizing agent uses more convenient; In described compound stabilizer, between each composition, mutually act synergistically,Thereby can long term storage; And can be widely used in various external diagnosis reagents, it is to reagentStablizing effect better, the term of validity that has effectively extended reagent.
Brief description of the drawings
Fig. 1 is that the compound stabilizer that embodiment 1 makes is measured reagent detection knot for creatine kinaseFruit c-t curve map.
Fig. 2 is that the compound stabilizer that embodiment 1 makes is measured reagent testing result for ureaC-t curve map.
Fig. 3 is that the compound stabilizer that embodiment 1 makes tries for determining alanine aminopheraseAgent testing result c-t curve map.
Fig. 4 is that the compound stabilizer that embodiment 2 makes is measured reagent detection knot for creatine kinaseFruit c-t curve map.
Fig. 5 is that the compound stabilizer that embodiment 2 makes is measured reagent testing result for ureaC-t curve map.
Fig. 6 is that the compound stabilizer that embodiment 2 makes tries for determining alanine aminopheraseAgent testing result c-t curve map.
Fig. 7 is that the compound stabilizer that embodiment 3 makes is measured reagent detection knot for creatine kinaseFruit c-t curve map.
Fig. 8 is that the compound stabilizer that embodiment 3 makes is measured reagent testing result for ureaC-t curve map.
Fig. 9 is that the compound stabilizer that embodiment 3 makes tries for determining alanine aminopheraseAgent testing result c-t curve map.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further elaborated, and these embodiment only doFor to explanation of the present invention, instead of for protection scope of the present invention is limited.
The reagent relating in following examples of the present invention has:
Bovine serum albumin(BSA), purity > 98%, BR, by the graceful bio tech ltd of upper HyponProduce;
Casein, BR, by Beijing, extensive and profound in meaning star biotechnology Co., Ltd produces;
Trehalose, purity > 98%, BR, by Jiangsu, Bao Lai bio tech ltd produces;
Propane diols, AR, is produced by Beijing Chemical Plant;
Polysorbas20, AR, is produced by Sigma;
Dithiothreitol (DTT), AR, is produced by Amresco;
Boratex, AR, Sigma produces;
Proclin950, IVD, Sigma produces;
Creatine kinase is measured reagent, urea is measured reagent and determining alanine aminopherase reagent, byOur company prepares voluntarily;
Quality-control product, is produced by Landau laboratory diagnosis Co., Ltd of Britain.
Embodiment 1
Compound stabilizer for external diagnosis reagent prepared by the present embodiment, it consists of:
The preparation method of described compound stabilizer is:
1) take respectively bovine serum albumin(BSA) 5g, casein 2g, two sulphur threoses by constituent contentAlcohol 0.8g, Boratex 3g and trehalose 10g, by above-mentioned each component in the ultra-pure water of 30mLFully dissolve, mix, obtain solution 1;
2) by constituent content measure respectively described propane diols 20mL, polysorbas20 2mL andProClin9501mL, and join in solution 1, mix, obtain solution 2;
3) solution 2 use ultra-pure waters are settled to 100mL, make described stable compositionAgent.
Embodiment 2
Compound stabilizer for external diagnosis reagent prepared by the present embodiment, it consists of:
Preparation method is with embodiment 1.
Embodiment 3
Compound stabilizer for external diagnosis reagent prepared by the present embodiment, it consists of:
Preparation method is with embodiment 1.
Experimental example 1
The compound stabilizer of the external diagnosis reagent respectively embodiment 1 being made joins creatineKinase assays reagent, urea are measured the R1 in reagent and determining alanine aminopherase reagentIn R2 reagent. Described compound stabilizer adds each in-vitro diagnosis examination by the amount of 10% (v/v)In agent. Get the each external diagnosis reagent dilute with water 10% that does not add compound stabilizer simultaneously(v/v) in contrast.
The quality-control product of described each external diagnosis reagent is:
Creatine kinase is measured reagent: Landau quality controlled serum level 2, creatine kinase target value 213U/L;Landau quality controlled serum level 3, creatine kinase target value 588U/L;
Urea is measured reagent: Landau quality controlled serum level 2, urea target value 7.16mmol/L; BrightRoad quality controlled serum level 3, urea target value 21.10mmol/L;
Determining alanine aminopherase reagent: Landau quality controlled serum level 2, alanine aminoTransferase target value 36U/L; Landau quality controlled serum level 3, ALT target value136U/L。
37 DEG C, 7 days accelerated stability tests:
1) external diagnosis reagent of getting 4 DEG C of storages is calibrated Biochemical Analyzer, and respectivelyDetect corresponding quality-control product, gained detected value is the detected value of the 0th day;
2) by 1) in external diagnosis reagent be divided into two groups, one group adds compound stabilizer, anotherOne group does not add compound stabilizer, and puts into 37 DEG C of insulating boxs simultaneously;
3) 1 day from putting into 37 DEG C of insulating boxs starts, and survey respectively every day on Biochemical AnalyzerDetermine quality-control product, until the 7th day, result is as shown in following table 1-3:
Table 1. creatine kinase is measured reagent testing result (unit: U/L)
Table 2. urea is measured reagent testing result (unit: mmol/L)
Table 3. determining alanine aminopherase reagent testing result (unit: U/L)
Experimental example 2
In this experimental example, except the compound stabilizer adding be embodiment 2 make, itsConsistent with experimental example 1 of remaining operation, within 37 DEG C, 7 days, accelerated stability test result is as following tableShown in 4-6:
Table 4. creatine kinase is measured reagent testing result (unit: U/L)
Table 5. urea is measured reagent testing result (unit: mmol/L)
Table 6. determining alanine aminopherase reagent testing result (unit: U/L)
Experimental example 3
In this experimental example, except the compound stabilizer adding be embodiment 3 make, itsConsistent with experimental example 1 of remaining operation, within 37 DEG C, 7 days, accelerated stability test result is as following tableShown in 7-9:
Table 7. creatine kinase is measured reagent testing result (unit: U/L)
Table 8. urea is measured reagent testing result (unit: mmol/L)
Table 9. determining alanine aminopherase reagent testing result (unit: U/L)
From the result of the test in experimental example 1 to 3, the reagent that does not add compound stabilizer along withThe passing of 37 DEG C of incubation times, the activity of reagent obviously declines, and has added embodiment 1,2Or reagent after the compound stabilizer making in 3 is along with the passing of 37 DEG C of incubation times, its examinationAgent is active decline very slow, especially in experimental example 2, after 37 DEG C, 7 days accelerated tests,Add the compound stabilizer that embodiment 2 makes each external diagnosis reagent activity still 90% withOn.
Claims (10)
1. for a compound stabilizer for external diagnosis reagent, it comprises sealer, surfaceActivating agent, little molecule organic reducing agent, nucleic acid compositions stabilizing agent, bioactivator protective agentAnd biological preservative; The content of described sealer is 1～55% (m/v), described surface-activeThe content of agent is 10.5～65% (v/v), the content of described little molecule organic reducing agent is 0.02～1% (m/v), the content of described nucleic acid compositions stabilizing agent is 0.3～4% (m/v), described lifeThe protectant content of active substances is 10～50% (m/v), the content of described biological preservativeBe 0.5～5% (v/v), surplus is ultra-pure water.
2. the compound stabilizer for external diagnosis reagent according to claim 1, itsBe characterised in that: the content of described sealer is 7～18% (m/v), described surfactantContent is 11～52% (v/v), and the content of described little molecule organic reducing agent is 0.03～0.8%(m/v), the content of described nucleic acid compositions stabilizing agent is 0.76～3% (m/v), described biologyThe protectant content of active material is 10～20% (m/v), and the content of described biological preservative is0.5～1% (v/v), surplus is ultra-pure water.
3. the compound stabilizer for external diagnosis reagent according to claim 2, itsBe characterised in that: described sealer is in bovine serum albumin(BSA), casein, gelatin and skimmed milk powerOne or more; Preferably, described sealer is bovine serum albumin(BSA) and casein, described inThe content of bovine serum albumin(BSA) is 10% (m/v), and described caseic content is 1% (m/v).
4. the compound stabilizer for external diagnosis reagent according to claim 2, itsBe characterised in that: described surfactant is in polysorbas20, polysorbate60, Tween 80 and propane diolsOne or more.
5. the compound stabilizer for external diagnosis reagent according to claim 4, itsBe characterised in that: described surfactant is polysorbas20 and propane diols, the content of described polysorbas20Be 2% (v/v), the content of described propane diols is 50% (v/v).
6. the compound stabilizer for external diagnosis reagent according to claim 2, itsBe characterised in that: described organic molecule reducing agent is dithiothreitol (DTT), its content is 0.03%(m/v)。
7. the compound stabilizer for external diagnosis reagent according to claim 2, itsBe characterised in that: described nucleic acid compositions stabilizing agent is Boratex, its content is 0.76% (m/v).
8. the compound stabilizer for external diagnosis reagent according to claim 2, itsBe characterised in that: described bioactivator protective agent is trehalose, its content is 20% (m/v).
9. the compound stabilizer for external diagnosis reagent according to claim 2, itsBe characterised in that: described biological preservative is ProClin950, its content is 1% (v/v).
10. a kit, its comprise in claim 1～9 described in any one forThe compound stabilizer of external diagnosis reagent.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106282308A (en) * | 2016-08-29 | 2017-01-04 | 湖南海源医疗科技有限公司 | A kind of in-vitro diagnosis biochemical reagents stabilizer |
| CN107942063A (en) * | 2016-10-13 | 2018-04-20 | 北京众驰伟业科技发展有限公司 | A kind of detection reagent of plasma fibrinogen, its detection method and application |
| CN108169152A (en) * | 2017-12-27 | 2018-06-15 | 山东博科生物产业有限公司 | A kind of angiotensin converting enzyme detection kit and its application method |
| CN108241058A (en) * | 2018-01-13 | 2018-07-03 | 中国医学科学院医学生物学研究所 | A kind of pre-coated detection method of III type D antigens of poliovirus and its detection kit and application |
| WO2019011125A1 (en) * | 2017-07-11 | 2019-01-17 | 深圳市伯劳特生物制品有限公司 | Composition for elisa kit and kit for detecting spectrum of helicobacter pylori antibody and preparation method thereof |
| CN111826417A (en) * | 2020-08-04 | 2020-10-27 | 武汉生之源生物科技股份有限公司 | N-acetyl-beta-D-glucosaminidase detection kit with good stability, preparation method and application |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106282308A (en) * | 2016-08-29 | 2017-01-04 | 湖南海源医疗科技有限公司 | A kind of in-vitro diagnosis biochemical reagents stabilizer |
| CN107942063A (en) * | 2016-10-13 | 2018-04-20 | 北京众驰伟业科技发展有限公司 | A kind of detection reagent of plasma fibrinogen, its detection method and application |
| WO2019011125A1 (en) * | 2017-07-11 | 2019-01-17 | 深圳市伯劳特生物制品有限公司 | Composition for elisa kit and kit for detecting spectrum of helicobacter pylori antibody and preparation method thereof |
| CN108169152A (en) * | 2017-12-27 | 2018-06-15 | 山东博科生物产业有限公司 | A kind of angiotensin converting enzyme detection kit and its application method |
| CN108241058A (en) * | 2018-01-13 | 2018-07-03 | 中国医学科学院医学生物学研究所 | A kind of pre-coated detection method of III type D antigens of poliovirus and its detection kit and application |
| CN111826417A (en) * | 2020-08-04 | 2020-10-27 | 武汉生之源生物科技股份有限公司 | N-acetyl-beta-D-glucosaminidase detection kit with good stability, preparation method and application |
| CN111826417B (en) * | 2020-08-04 | 2023-01-17 | 武汉生之源生物科技股份有限公司 | N-acetyl-beta-D-glucosaminidase detection kit with good stability, preparation method and application |
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