CN106350499A - Stabilizer of thrombin solution - Google Patents
Stabilizer of thrombin solution Download PDFInfo
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- CN106350499A CN106350499A CN201610872528.7A CN201610872528A CN106350499A CN 106350499 A CN106350499 A CN 106350499A CN 201610872528 A CN201610872528 A CN 201610872528A CN 106350499 A CN106350499 A CN 106350499A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
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Abstract
The invention discloses a stabilizer of a thrombin solution, a thrombin solution and a relevant detection reagent, a method for preparing the stable thrombin solution and application of the stabilizer of the thrombin solution.
Description
Technical field
The present invention relates to enzyme industrial circle and field of immunodetection, relate in particular to thrombin solution stabilizer,
The detectable of thrombin solution and correlation, prepare the method for stable thrombin solution and the stabilizer of thrombin solution
Purposes.
Background technology
Fibrinogen (fibrinogen, fib) is the fibrinous precursor of the main protein as blood clot
Matter, is produced by hepatic parenchymal cellses.About 80% of Fibrinogen in organism is distributed in blood plasma and (is about in normal adult
200～400mg/dl), remaining is distributed in tissue.Fibrinogen be containing through disulfide-bonded 3 pairs of polypeptides (a α, b β and
γ) the glycoprotein of chain, a α, b β chain is decomposed by thrombin and forms fibrin, thus playing, thrombosis generate, stop blooding blood
The Main Function of bolt.Clinically, increase in inflammation, reduce in the hepatic insufficiency of height, dic etc..
The clinical meaning of fibrinogen assay includes but is not limited to: 1. pathologic increases: (1) Pre-thrombosis State and thrombosis
Property disease when, body coagulation function strengthens, and plasma fibrinogen increases, such as acute myocardial infarction, diabetes, gestational hypertension
Disease, atherosclerosiss, malignant tumor etc..(2) albumen synthesis increases, such as connective tissue disease, multiple myeloma etc..(3) anti-
Answering property increases, such as actute infection, acute nephritiss, burn, shock, after major operation etc..2. pathologic reduces: (1) consumes excessively, leads
Plasma content is caused to reduce, such as dic etc..(2) Fibrinolytic Activities strengthen, and fib is decomposed, such as constitutional hyperfibrinolysiss disease etc..(3)
Synthesis reduces, such as hepatitis gravis, liver cirrhosis etc..
The algoscopy of Fibrinogen includes thrombin additive process (clauss method), Clotting-time method (pt algorithm), exempts from
Epidemic disease turbidimetry (tia), emulsion technique etc., typically adopt thrombin additive process.
Under thrombin additive process, the content of Fibrinogen ultimately forms fibre according to Fibrinogen and thrombin action
The principle of fibrillarin measures, and makes standard curve with international standard substance for reference blood plasma, during with thrombin to measure the clotting of plasma
Between, i.e. thrombin time (thrombin time, tt), gained PCT and fibrinogen concentration in blood plasma are in negative
Correlation, thus obtain the content of Fibrinogen.
Specifically, thrombin time refers to add the time of the clotting of plasma after standardized thrombinogen in blood plasma.
Under thrombin action, the Fibrinogen in blood plasma to be checked is changed into fibrin.When anticoagulant substanceses in blood plasma to be checked
When increasing, thrombin time extends.When blood plasma to be checked be in acid, test object suffers from abnormal fibrous proteinemia situations such as
Under, thrombin time shortens.
For example, thrombin time prolongation sees plasma fibrinogen and lowers or textural anomaly；Clinical practice heparin, or
Heparin sample anticoagulant substanceses when hepatopathy, nephropathy and systemic lupus erythematosus (sle) increase；Fibrinolytic systemic-function is hyperfunction.
The thrombin using in fibrinogen assay, cuts peptide from the thrombinogen as its precursor and forms tool
There is the α-thrombin of coagulation activity.Known this α-thrombin selfdecomposition can become the β without coagulation activity of low-molecular-weight-solidifying
Hemase, and then resolve into γ-thrombin, when preserving for a long time, generally carry out lyophilization.
For measuring the reagent of Fibrinogen (fib) content or thrombin time (tt), existing reagent is
Freeze-dried reagent, except expensive, in use, is required to redissolve, in-convenience in use, and will necessarily bring and redissolve
Cause volumetric errors in journey, lead to result that deviation occurs.And there is not this problem in liquid reagent and easy to use, that is, open
Use, difference between batch is less, result is more accurate.Therefore urgently need to develop the stabilizer that can stablize thrombin solution and
Stable thrombin solution product.
Content of the invention
The technical problem to be solved is the defect being not easy to preservation for thrombin solution, improves thrombin molten
The stability of liquid, thus extending the holding time, reducing and preserving cost, and convenient use reduces experimental error.
Present inventors discovered unexpectedly that lactate ion and glutamate ion have stable thrombin solution
Cooperative effect.
The invention provides for the stabilizer of thrombin solution, it comprises water-soluble lactic acid compound and water solublity paddy ammonia
Acid compound.In one embodiment, described stabilizer is by water-soluble lactic acid compound and water solublity glutamic acid compounds group
Become.In another embodiment, described stabilizer comprises water-soluble lactic acid compound, water solublity glutamic acid compounds and molten
Agent.In another embodiment in addition, described stabilizer comprises water-soluble lactic acid compound, water solublity glutamic acid chemical combination
Thing, solvent and those skilled in the art being capable of conventional use of auxiliary element (such as buffer agent, surfactant, preservative
Deng).Described solvent can be water, organic solvent or those skilled in the art can conventional determine or the solvent that uses in one
Plant or two or more combinations.Preferably, described solvent is water.
Preferably, in the stabilizer for thrombin solution of the present invention, water-soluble lactic acid compound and water solublity paddy ammonia
The ratio of acid compound is 1:50 to 50:1.Preferably, the upper limit of described ratio can be 40:1,30:1,20:1,10:1,9:
1st, 8:1,7:1,6:1,5:1,4:1,3:1,2:1, the lower limit of described ratio can be 1:40,1:30,1:20,1:10,1:9,1:
8th, 1:7,1:6,1:5,1:4,1:3,1:2, described ratio can be above-mentioned higher limit and the scope of lower limit combination in any.More excellent
Selection of land, described ratio can be 1:1.
Preferably, described water-soluble lactic acid compound can be water-soluble lactic acid salt.Described water-soluble lactic acid salt can be
One of calcium lactate, EINECS 212-761-8, magnesium lactate or zinc lactate or two or more combinations.Preferably, described water-soluble lactic acid salt
It is calcium lactate, EINECS 212-761-8 or a combination thereof.Most preferably, described water-soluble lactic acid salt is calcium lactate or EINECS 212-761-8.
Water-soluble lactic acid compound used in the present invention is commercially available, for example, be derived from Shanghai Aladdin biochemical technology stock
The trade mark of part company limited (Shanghai Jing Chun biochemical technology limited company) be the calcium lactate of Aladdin (aladdin) (for example,
Article No. c110506), from Sigma-Aldrich Co., Ltd (sigma-aldrich llc) calcium lactate (for example,
Production code member 21175), from Chemical Reagent Co., Ltd., Sinopharm Group EINECS 212-761-8 etc..
Preferably, described water solublity glutamic acid compounds can be water-soluble glutamate.It is highly preferred that described water solublity
Glutamate, Glu is sodium glutamate, Kaglutam or a combination thereof.Most preferably, described water-soluble glutamate is sodium glutamate.
Water solublity glutamic acid compounds used in the present invention are commercially available, for example, have from Shanghai sky biological reagent
The sodium glutamate of limit company, the trade mark from Shanghai Yuan Ye bio tech ltd are Kaglutam (for example, the article No. of source leaf
S20427) etc..
Present invention also offers comprising the thrombin solution of the stabilizer of the present invention.
The thrombin solution (referred to as " thrombin solution of the present invention ") that the stabilizer of the present invention is suitable for is to make thrombin
It is suspended or dissolved in the mixing of the aqueous solution, organic solvent solution or water and organic solvent in water and/or organic solvent
Solution, preferably aqueous solution or water and organic solvent mixed solution.Described organic solvent, as long as do not result in dividing of thrombin
Solution and activity suppression etc., and its final use is not affected, just it is not particularly limited, for example dimethyl sulfoxide, glycerol, poly- second
Glycol, polypropylene glycol etc..By one of these materials or can also be used in combination.
Preferably, in the thrombin solution of the present invention, calculated with the cumulative volume of thrombin solution, water-soluble lactic acid compound
Concentration be 1g/l to 20g/l；It is highly preferred that in the thrombin solution of the present invention, being calculated with the cumulative volume of thrombin solution, water
The concentration of dissolubility polylactides is 2g/l to 10g/l；It is highly preferred that in the thrombin solution of the present invention, with thrombin solution
Cumulative volume calculate, the concentration of water-soluble lactic acid compound is 2g/l, 3g/l, 4g/l, 5g/l, 6g/l, 7g/l, 8g/l, 9g/l
Or 10g/l.It is further preferred that in the thrombin solution of the present invention, being calculated with the cumulative volume of thrombin solution, water-soluble lactic acid
The concentration of compound is 7.7g/l.
Preferably, in the thrombin solution of the present invention, calculated with the cumulative volume of thrombin solution, water solublity glutamic acid chemical combination
The concentration of thing is 1g/l to 150g/l；It is highly preferred that in the thrombin solution of the present invention, with the total volume meter of thrombin solution
Calculate, the concentration of water solublity glutamic acid compounds is 10g/l to 100g/l；It is highly preferred that in the thrombin solution of the present invention, with solidifying
The cumulative volume of hemase solution calculates, the concentration of water solublity glutamic acid compounds be 10g/l, 15g/l, 20g/l, 25g/l, 30g/l,
35g/l、40g/l、45g/l、50g/l、55g/l、60g/l、65g/l、70g/l、75g/l、80g/l、85g/l、90g/l、95g/l
Or 100g/l；It is further preferred that in the thrombin solution of the present invention, being calculated with the cumulative volume of thrombin solution, water solublity paddy
The concentration of propylhomoserin compound is 50g/l.
The thrombin solution of the present invention can also comprise buffer agent, surfactant and preservative.
Preferably, described buffer agent can be 4- hydroxyethyl piperazine ethanesulfonic acid (n- (2- ethoxy) piperazine-n'-2- ethane
Sulfonic acid；Molecular formula: c8h18n2o4s, also known as hepes acid), citric acid, phosphoric acid, acetic acid, imidazoles, 3- (n- morpholine) the third sulphur
(mops), two (2- ethoxy) imido grpup three (methylol) methane (bis-tris), trishydroxymethylaminomethane (tris), 3-
(n- morpholinyl) -2- hydroxy-propanesulfonic acid (mopso), n- (2- acetylamino)-imino-diacetic acetic acid (ada) or MES
One of or two or more combinations (mes)；It is highly preferred that buffer agent is 4- hydroxyethyl piperazine ethanesulfonic acid.
Buffer agent used in the present invention is commercially available, for example, be derived from the 4- of Suzhou City Bake bio tech ltd
Hydroxyethyl piperazine ethanesulfonic acid, 3- (n- the morpholinyl) -2- hydroxyl third from the uncommon love of ladder (Shanghai) chemical conversion industry Development Co., Ltd
Sulfonic acid (for example, product coding h0671), the trade mark from Sa En chemical technology (Shanghai) Co., Ltd. are the 2- morpholine of An Naiji
Ethyl sulfonic acid (for example, goods number d090002).
As long as the addition of described buffer agent has the amount of buffer capacity with regard to there is no particular limitation, people in the art
Member can the conventional content determining the buffer agent being suitable for using.But, the inventors found that the thrombin of the present invention is molten
In liquid, calculated with the cumulative volume of thrombin solution, when the concentration of buffer agent is values below scope, be more beneficial for realizing this
Bright purpose: preferably, in the thrombin solution of the present invention, calculated with the cumulative volume of thrombin solution, the concentration of buffer agent is
1g/l to 50g/l；It is highly preferred that in the thrombin solution of the present invention, calculated with the cumulative volume of thrombin solution, buffer agent dense
Degree is 5g/l to 24g/l；It is highly preferred that in the thrombin solution of the present invention, being calculated with the cumulative volume of thrombin solution, buffer agent
Concentration be 5.9g/l to 23.8g/l；It is highly preferred that in the thrombin solution of the present invention, with the total volume meter of thrombin solution
Calculate, the concentration of buffer agent be 5.9g/l, 6g/l, 7g/l, 8g/l, 9g/l, 10g/l, 11g/l, 11.9g/l, 12g/l, 13g/l,
14g/l, 15g/l, 16g/l, 17g/l, 17.9g/l, 18g/l, 19g/l, 20g/l, 21g/l, 22g/l, 23g/l or 23.8g/l；
It is further preferred that in the thrombin solution of the present invention, being calculated with the cumulative volume of thrombin solution, the concentration of buffer agent is
11.9g/l.
Described surfactant can be anionic surfactant, cationic surfactant, amphion table
One of face activating agent or nonionic surfactant or two or more combinations.
Described anionic surfactant can be sodium lauryl sulphate, dodecyl sodium sulfate, dodecyl-n-
One of sodium sarcosinate, sodium cholate, NaTDC or sodium taurodeoxycholate or two or more combinations.
Described cationic surfactant can be cetyl trimethylammonium bromide, four decyl ammonium bromide or dodecane
One of pyridinum chloride or two or more combinations.
Described zwitterionic surfactant can be 3- [(3- gallbladder amido propyl) Dimethyl Ammonium] -1- propane sulfonic acid salt, 3-
[(3- gallbladder amido propyl) Dimethyl Ammonium] -2- hydroxyl -1- propane sulfonic acid salt, palmityl LYSOLECITHIN SUNLECITHIN A, dodecyl-n- glycine betaine
Or one of dodecyl-Beta-alanine or two or more combinations.
Described nonionic surfactant can be octyl glucoside, heptyl glucosinolate, capryl-n- methyl Portugal
Osamine, polyoxyethylene lauryl ether, polyoxyethylene seven methylhexyl ether, Triton X100, polyoxyethylene nonyl
One of base phenyl ether, polyoxyethylene fatty acid ester, sucrose fatty acid ester or polyoxyethylene sorbitol ester or two kinds with
On combination.
Preferably, described surfactant is nonionic surfactant；It is highly preferred that described surfactant is poly-
Oxygen ethylene Arlacel-80 (also known as Tween 80).
Surfactant used in the present invention is commercially available, for example, be derived from the tween of Chengdu Ke Long chemical reagent factory
80th, be derived from Shanghai Aladdin biochemical technology limited company (Shanghai Jing Chun biochemical technology limited company) trade mark be Ah
The Tween 80 (for example, article No. t104866) of Latin (aladdin), the trade mark of Shanghai Yuan Ye bio tech ltd are source leaf
Sucrose fatty acid ester (for example, article No. s30894) etc..
As long as the addition of described surfactant makes the stability-enhanced amount of thrombin solution, not especially
Limit, those skilled in the art can the conventional content determining the surfactant being suitable for using.But, the present inventor
Find, in the thrombin solution of the present invention, to calculate with the cumulative volume of thrombin solution, when the concentration of surfactant is following number
During value scope, it is more beneficial for realizing the purpose of the present invention: preferably, calculate with the cumulative volume of thrombin solution, surfactant
Concentration be 10 μ l/l to 1000 μ l/l；It is highly preferred that being calculated with the cumulative volume of thrombin solution, the concentration of surfactant is
50 μ l/l to 500 μ l/l；It is highly preferred that being calculated with the cumulative volume of thrombin solution, the concentration of surfactant be 100 μ l/l extremely
400μl/l；It is further preferred that being calculated with the cumulative volume of thrombin solution, the concentration of surfactant is 100 μ l/l, 200 μ
L/l, 300 μ l/l or 400 μ l/l；It is further preferred that being calculated with the cumulative volume of thrombin solution, the concentration of surfactant
It is 200 μ l/l.
Described preservative can be sodium azide (nan3), Ciprofloxacin, one of propanoic acid or sodium benzoate or two kinds with
On combination.Preferably, described preservative is sodium azide.
Preservative used in the present invention is commercially available, for example, come precious biotechnology (Shanghai) limited company westerly
Trade mark be the sodium azide (for example, article No. be 0639) of amresco, be derived from Chengdu Hua Xia chemical reagent company limited sodium azide
(the brilliant pure biochemical technology share in Shanghai is limited for (for example, article No. is h1100046), Shanghai Aladdin biochemical technology limited company
Company) trade mark be the sodium benzoate (for example, article No. be s104128) of Aladdin (aladdin), be derived from Sigma-Aldrich
(for example, production code member is the preservative for proclin300 for the trade mark of Co., Ltd (sigma-aldrich llc)
48914-u) etc..
As long as just there is no particular limitation in prescribed limit for the addition of described preservative.Those skilled in the art are permissible
The conventional content determining the preservative being suitable for using.But, the inventors found that in the thrombin solution of the present invention, with
The cumulative volume of thrombin solution calculates, and when the concentration of preservative is values below scope, is more beneficial for realizing the mesh of the present invention
: preferably, calculated with the cumulative volume of thrombin solution, the concentration of preservative is 0.1g/l to 5.0g/l；It is highly preferred that with solidifying
The cumulative volume of hemase solution calculates, and the concentration of preservative is 0.2g/l to 2.5g/l；It is highly preferred that it is overall with thrombin solution
Long-pending calculating, the concentration of preservative is 0.4g/l to 1.3g/l；It is further preferred that being calculated with the cumulative volume of thrombin solution, prevent
The concentration of rotten agent be 0.4g/l, 0.5g/l, 0.6g/l, 0.7g/l, 0.8g/l, 0.9g/l, 1.0g/l, 1.1g/l, 1.2g/l or
1.3g/l；It is further preferred that being calculated with the cumulative volume of thrombin solution, the concentration of preservative is 1.0g/l.
Used in the present invention, thrombin can be the thrombin of the animal origins such as people, pig, cattle, pass through genetic engineering legal system
Standby thrombin or commercially available medicine.Thrombin used in the present invention is commercially available, is e.g. derived from Hunan one lattice pharmacy
The thrombin lyophilized powder of company limited, coagulating from Sigma-Aldrich Co., Ltd (sigma-aldrich llc)
Hemase preparation (for example, production code member is 10602400001) etc..
As long as it is just not special that the addition of the thrombin in the thrombin solution of the present invention is suitable for the purpose of the present invention
Limit.It was found by the inventors of the present invention that in the thrombin solution of the present invention, being calculated with the cumulative volume of thrombin solution, thrombin
Concentration can be 1ku/l to 120ku/l；It is highly preferred that in the thrombin solution of the present invention, with the cumulative volume of thrombin solution
Calculate, the concentration of thrombin can be 1ku/l, 2ku/l, 3ku/l, 4ku/l, 5ku/l, 6ku/l, 7ku/l, 8ku/l, 9ku/l,
10ku/l、20ku/l、30ku/l、40ku/l、50ku/l、60ku/l、70ku/l、80ku/l、90ku/l、100ku/l、110ku/
L or 120ku/l；It is highly preferred that in the thrombin solution of the present invention, calculated with the cumulative volume of thrombin solution, thrombin dense
Degree can be 2ku/l to 110ku/l, 5ku/l to 100ku/l, 10ku/l to 90ku/l, 15ku/l to 80ku/l, 20ku/l extremely
70ku/l, 25ku/l to 60ku/l, 30ku/l to 50ku/l；It is further preferred that in the thrombin solution of the present invention, with blood coagulation
The cumulative volume of enzymatic solution calculates, and the concentration of thrombin can be 2ku/l, 4ku/l, 30ku/l, 70ku/l, 120ku/l.
Preferably, the thrombin solution of the present invention, is calculated with the cumulative volume of thrombin solution, comprising concentration is 7.7g/l
Calcium lactate, concentration be the sodium glutamate of 50g/l, concentration be the 4- hydroxyethyl piperazine ethanesulfonic acid of 11.9g/l, concentration be 1.0g/l
Sodium azide, concentration are the polyoxyethylene sorbitan monooleate dehydration of 200 μ l/l.It is highly preferred that in above-mentioned thrombin solution, bag
It is the thrombin of 2ku/l, 4ku/l, 30ku/l or 70ku/l containing concentration.
Preferably, the thrombin solution of the present invention, is calculated with the cumulative volume of thrombin solution, comprising concentration is 7.7g/l
EINECS 212-761-8, concentration be the sodium glutamate of 50g/l, concentration be the 4- hydroxyethyl piperazine ethanesulfonic acid of 11.9g/l, concentration be 1.0g/l
Sodium azide, concentration are the polyoxyethylene sorbitan monooleate dehydration of 200 μ l/l.It is highly preferred that in above-mentioned thrombin solution, bag
It is the thrombin of 2ku/l, 4ku/l, 30ku/l or 70ku/l containing concentration.
Present invention also offers the method preparing thrombin solution, methods described includes the step of the stabilizer using the present invention
Suddenly.
Present invention also offers the stabilizer of the present invention is used for improving the purposes of thrombin solution stability.
Present invention also offers the thrombin solution of the stabilizer of the present invention or the present invention is used for the purposes of reagent preparation.
Present invention also offers a kind of reagent, it contains the thrombin solution of the stabilizer of the present invention or the present invention.
Stabilizer or the reagent of thrombin solution preparation of the present invention or stablizing containing the present invention using the present invention
The reagent of the thrombin solution of agent or the present invention can be referred to as the reagent of the present invention.
Preferably, described reagent is the reagent for detection fibers proteinogen or thrombin time.
Preferably for the present invention for detection fibers proteinogen reagent, the containing of the thrombin in thrombin solution
Measure as 30ku/l or 70ku/l；Thrombin for the present invention for detecting the reagent of thrombin time, in thrombin solution
Content be 2ku/l or 4ku/l.
Present invention also offers a kind of test kit, it comprises the reagent of the present invention.
The present inventors have noted that, in the case that thrombin solution other components are constant, under different concentration of thrombin, contain
The measured value having the reagent of the thrombin solution of the present invention can overall improve, but this is to the thrombin solution of the present invention and correlation
The purposes of the stability of product itself and correlation, the enforcement of method do not have an impact, and the raising of measured value also will not hinder this
The realization of the function of bright thrombin solution and Related product.In order to obtain the measured value in ideal range, people in the art
Member can conventional adjustment thrombin content realizing above-mentioned target.
The thrombin solution convenient use of the stabilizer containing the present invention, need not redissolve, it is to avoid due to redissolving volume difference
Lead to error.Plug and play, difference between batch is less, and result is more accurate.
Thrombin solution stability using the stabilizer preparation of the present invention is strong, stablizes more than 1 year for 2-8 DEG C, 37 DEG C stable
More than 20 days, 2-8 DEG C opened stability more than 10 days.Thus, the stabilizer of the present invention can improve stablizing of thrombin solution
Property, improve storage form and the condition of Related product, reduce the cost producing and applying.
Specific embodiment
Used in the following examples of the present invention, to be respectively as follows: Shanghai Aladdin biochemical technology share limited for each Component Source
The trade mark of company (Shanghai Jing Chun biochemical technology limited company) is the calcium lactate (article No. of Aladdin (aladdin)
C110506), it is derived from the EINECS 212-761-8 of Chemical Reagent Co., Ltd., Sinopharm Group, be derived from Shanghai Yuan Ye bio tech ltd
Trade mark is the Kaglutam (for example, article No. s20427) of source leaf, the sodium glutamate of Shanghai sky biological reagent company limited, Suzhou
The 4- hydroxyethyl piperazine ethanesulfonic acid (hepes acid) of city Bake bio tech ltd, the tween of Chengdu Ke Long chemical reagent factory
80th, the trade mark of west treasured biotechnology (Shanghai) limited company is the sodium azide (article No. 0639) of amresco, Hunan one lattice system
The thrombin lyophilized powder of medicine company limited.
Embodiment 1
The preparation of thrombin time (tt) detectable of the stabilizer containing the present invention
Weigh hepes acid 11.9g, calcium lactate 7.7g, sodium glutamate 50g, sodium azide 1g, Tween 80 0.2g, add distillation
Dissolve in water, adjust ph to 6.4 with sodium hydroxide, be settled to 1l.Take thrombin 4 (1000u/ props up), prepare buffering with above
Liquid 1ml/ props up dissolving, takes dissolving thrombin 3.6ml (scalable enzyme amount controls difference between batch), adds in 1l buffer and shake up, tt examines
Test agent is prepared and is completed.
Product performance index
1. repeatability: the coefficient of variation (cv)≤5%；With normal Quality Control blood plasma retest 10 times, calculate measure average
ValueWith standard deviation (s).Calculate the coefficient of variation (cv) according to formula (1).Acquired results should meet wanting of following 3. stability
Ask.
In formula:
In s: standard deviation
Measure average
2. difference between batch: the coefficient of variation (cv)≤10%；Test the reagent of 3 different batches with normal Quality Control blood plasma respectively,
Each batch measures retest 10 times, calculates 30 measured value meansigma methodssWith standard deviation (s), calculate according to formula (1)
The coefficient of variation (cv).
3. stability
2 DEG C～8 DEG C airtight preservations of this product, are newly opened one bottle of mensure Quality Control blood plasma every time, were contrasted with first day, relative deviation exists
In 15%, 12 months effect duration.
Embodiment 2
The detection method of thrombin time (tt) detectable
Take blood plasma 100 μ l, 37 DEG C preheat 2 minutes, are subsequently adding thrombin reagent 100 μ l, in ca550 full automatic blood-coagulation instrument
(producer sysmex) measures according to the description of producer.
Points for attention:
1. blood sampling should avoid haemolysis, prevents tissue fluid to be mixed into.Will smoothly during blood drawing, anticoagulant fully will can not have sludged blood；
Platelet must be removed during separated plasma.
2. anticoagulant should not be made with edta and heparin during sample collection.The ratio of blood and anticoagulant should accurately, blood sampling volume
The specimen that deviation is more than 10% should not use.
If 3. hematocrit≤20% or >=55%, need to adjust the ratio of blood sample and anticoagulant, anticoagulant milliliter number
=0.00185 × blood milliliter number × (100- hematocrit).
4. use calibrated after measuring pipette and sample injector.
5. use the plastics of cleanliness without any pollution or the glass drying oven of silication.
6. experimental temperature controls at 37 DEG C ± 1.0 DEG C.
7. reagent and sample size can change as needed in proportion.
8. reagent, using finishing, should be wiped clean bottleneck, screw bottle cap as early as possible, put back in time and protect under the condition of storage of requirement
Deposit.
Embodiment 3
The stability experiment of thrombin time (tt) detectable of the stabilizer containing the present invention
Scheme according to embodiment 1 prepares thrombin time (tt) detectable, using ca550 full automatic blood-coagulation instrument (factory
Family sysmex) according to the description of producer measure following under the conditions of described reagent stability
1.37 DEG C accelerating the failure.
2.2-8 DEG C corkage stability.
3.2-8 DEG C of long-time stability.
37 DEG C surely accelerate the failure, normal Quality Control blood plasma measured value, stable within measured value January.It is shown in Table 1.
1 37 DEG C of table accelerates heat damage experiment
Corkage stability experiment, is put in 2-8 DEG C, 15 days, measured value was more stable after corkage.It is shown in Table 2.
2-8 DEG C of corkage stability of table 2
2-8 DEG C of long-time stability, 15 months, measured value was more stable.It is shown in Table 3.
Table 3 long-time stability
Embodiment 4
The preparation of Fibrinogen (fib) detectable of the stabilizer containing the present invention
(1) reagent preparation: weigh hepes acid 11.9g, calcium lactate 7.7g, sodium glutamate 50g, sodium azide 1g, Tween 80
0.2g, adds in distilled water and dissolves, and adjusts ph to 6.4 with sodium hydroxide, is settled to 1l.Take thrombin 70 (1000u/ props up),
Prop up dissolving with the above buffer 1ml/ for preparing, all add in 1l buffer and shake up, fib detectable is prepared and completed.
(2) prepared and diluted sample buffer: weigh imidazoles 3.06g, sodium chloride 5.22g, sodium citrate 1.2g, sodium azide
0.95g, adds in distilled water and dissolves, and adjusts ph to 7.30 with sodium hydroxide, is settled to 1l.After stable in two days, adjust ph again
To 7.30.
Product performance index
1. accuracy: take reference material or the test of accurateness Quality Control thing, in triplicate, obtain meansigma methodss, according to formula (2) meter
Calculate the relative deviation of meansigma methodss and reference material or main calibration object, result should meet relative deviation should wanting in the range of ± 15%
Ask.Computing formula:
In formula:
X --- detection average；
Xc --- reference material or accurateness Quality Control thing sign value.
2. repeatability
Distinguish retest 10 times with normal Quality Control blood plasma and abnormal Quality Control blood plasma, and calculate the meansigma methodss of mensureWith
Standard deviation (s).Calculate the coefficient of variation (cv) by formula (1), result should meet the requirement of the coefficient of variation (cv)≤8%.
3. difference between batch
Test the reagent of 3 different batches with normal Quality Control blood plasma, each batch is tested 10 times, calculate 30 measured values and put down
AverageWith standard deviation (s), calculate the coefficient of variation (cv) according to formula (1), result should meet the coefficient of variation (cv)≤15%
Requirement.
4. stability
2 DEG C～8 DEG C airtight preservations of this product, newly open every time one bottle mensure Quality Control blood plasma, relative deviation in 15%, effect duration
12 months.
Embodiment 5
The detection method of Fibrinogen (fib) detectable
(1) standard curve is set up
Dilute reference blood plasma with diluent according to the form below, you can acquisition series concentration:
Take fib detectable pre-temperature 3 minutes, to 37 DEG C.Take the reference blood plasma after dilution 100 μ l, 37 DEG C of pre-temperatures 2 minutes.
Add 50 μ l pre-temperature reagent, mix at once and timing, record setting time.With the obtained target level of reference blood plasma gradient dilution it is
Abscissa, corresponding setting time (second) is vertical coordinate, and measurement result is mapped on double logarithmic curve, or input instrument is according to corresponding
Mathematical model automatically generate working curve.
(2) sample measures
Sample dilution buffer carries out 10 times of dilutions, and determination step is with the preparation of working curve.Automatically coagulated with ca550
Blood instrument (producer sysmex) measures according to the description of producer.
Points for attention:
1. blood sampling should avoid haemolysis, prevents tissue fluid to be mixed into.Will smoothly during blood drawing, anticoagulant fully will can not have sludged blood；
Platelet must be removed during separated plasma.
2. anticoagulant should not be made with edta and heparin during sample collection.The ratio of blood and anticoagulant should accurately, blood sampling volume
The specimen that deviation is more than 10% should not use.
If 3. hematocrit≤20% or >=55%, need to adjust the ratio of blood sample and anticoagulant, anticoagulant milliliter number
=0.00185 × blood milliliter number × (100- hematocrit).
4. use calibrated after measuring pipette and sample injector.
5. use the plastics of cleanliness without any pollution or the glass drying oven of silication.
6. experimental temperature controls at 37 DEG C ± 1.0 DEG C.
7. reagent and sample size can change as needed in proportion.
8. reagent, using finishing, should be wiped clean bottleneck, screw bottle cap as early as possible, put back in time and protect under the condition of storage of requirement
Deposit.
Embodiment 6
The stability experiment of Fibrinogen (fib) detectable of the stabilizer containing the present invention
Scheme according to embodiment 4 prepares Fibrinogen (fib) detectable, using ca550 full automatic blood-coagulation instrument (factory
Family sysmex) according to the description of producer measure following under the conditions of described reagent stability.
1. 37 DEG C accelerate the failure.
2. 2-8 DEG C of corkage stability.
3. 2-8 DEG C of long-time stability.
37 DEG C accelerate the failure, normal Quality Control blood plasma measured value, stable within measured value January.It is shown in Table 4.
4 37 DEG C of table accelerates heat damage experiment
Corkage stability experiment, is put in 2-8 DEG C after corkage, open 17 days, measured value is more stable.It is shown in Table 5.
2-8 DEG C of corkage stability of table 5
2-8 DEG C of long-time stability, 416 days, measured value was more stable.It is shown in Table 6.
2-8 DEG C of long-time stability of table 6
Embodiment 7
There is the cooperative effect stablizing thrombin solution between water-soluble lactic acid compound and water solublity glutamic acid compounds
Under conditions of its dependent variable is consistent, for containing only calcium lactate, contain only sodium glutamate and contain breast simultaneously
Stabilization time at 37 DEG C of three kinds of thrombin solutions mensure of sour calcium and sodium glutamate, with relative deviation 10% as nature controlling line, see
Examine the testing result of above-mentioned thrombin solution.Testing result takes the average of 5 tests.
The application also measures the stabilization time at 37 DEG C containing calcium lactate and Kaglutam thrombin solution simultaneously, with
Relative deviation 10% is nature controlling line, observes the testing result of above-mentioned thrombin solution.Testing result takes the average of 3 tests.
In thrombin solution in sequence number 1-4 in table 7, the content of each composition is respectively as follows: calcium lactate 7.7g/l (such as containing), paddy
Propylhomoserin sodium 50g/l (such as containing), Kaglutam 50g/l (such as containing), hepes acid 11.9g/l, sodium azide 1g/l, Tween 80
200 μ l/l, thrombin 4ku/l (solution of sequence number 1-3) or 2ku/l (solution of sequence number 4).
Table 7: calcium lactate and water solublity glutamic acid compounds stablize the cooperative effect of thrombin solution
Table 8a calcium lactate and water solublity glutamic acid compounds stablize the test of thrombin solution
From table 7, table 8a it is observed that calcium lactate and water solublity glutamic acid compounds simultaneously in the presence of, thrombin solution
Stable natural law the longest.Therefore, there is the collaborative effect stablizing thrombin solution between calcium lactate and water solublity glutamic acid compounds
Should.
The present invention also have chosen the thrombin solution containing EINECS 212-761-8 and sodium glutamate to verify water-soluble lactic acid further
There is the cooperative effect stablizing thrombin solution between compound and water solublity glutamic acid compounds.Measure 37 DEG C at stable when
Between, with relative deviation 10% as nature controlling line, observe the testing result of above-mentioned thrombin solution.Testing result takes the equal of 3 tests
Value.
Above-mentioned EINECS 212-761-8 and sodium glutamate are respectively as follows: EINECS 212-761-8 as the content of composition each in the thrombin solution of stabilizer
7.7g/l, sodium glutamate 50g/l, hepes acid 11.9g/l, sodium azide 1g/l, Tween 80 200 μ l/l, thrombin 30ku/l
(fib reagent) or 2ku/l (tt reagent).The stability test of above-mentioned thrombin solution is as follows:
Table 8b EINECS 212-761-8 and sodium glutamate stablize the test of thrombin solution
From table 8b, EINECS 212-761-8 and sodium glutamate also have good synergisticing stable effect.
Therefore, can be seen that from above-mentioned experiment and exist between water-soluble lactic acid compound and water solublity glutamic acid compounds
Stablize the cooperative effect of thrombin solution.
Embodiment 8
Having or not sodium glutamate affects Experimental comparison to tt reagent stability.
According to the thrombin solution formula in embodiment 1, in the case of other components content is constant, at measuring 37 DEG C respectively
Stability containing sodium glutamate and the no thrombin solution of sodium glutamate.
9 37 DEG C of table has or not sodium glutamate impact
No paddy ammonia is substantially better than by the formulation stability that the result of the test of table 9 can be seen that containing sodium glutamate and calcium lactate
The stability of the formula of sour sodium.
Embodiment 9
The stablizing effect to thrombin solution for the aminoacid or its salt of calcium lactate and other non-glutamic acids
In thrombin solution, the content of each composition is respectively as follows: calcium lactate 7.7g/l, glycine 50g/l (such as containing), smart ammonia
Sour 50g/l (such as containing), serine 50g/l (such as containing), glutamic acid 50g/l (such as containing), hepes acid 11.9g/l, sodium azide
1g/l, Tween 80 200 μ l/l.In tt reagent, thrombin is 4ku/l, and in fib reagent, thrombin is 70ku/l.
With relative deviation 10% as nature controlling line, according to the experiment knot of table 10a in table 7 in embodiment 7 and the present embodiment, 11
Really, calcium lactate and the aminoacid of other non-glutamic acids or its salt do not enable water solublity glutamic acid compounds to thrombin solution
Stablizing effect.
The aminoacid of table 10a: calcium lactate and other non-glutamic acids stablizes the cooperative effect of thrombin solution
Table 10b: calcium lactate stablizes the cooperative effect of thrombin solution with glycine or L-Valine
In embodiment 10-14, in thrombin solution during a certain component content change, the content of other compositions follows following number
Value: calcium lactate 7.7g/l, sodium glutamate 50g/l, hepes acid 11.9g/l, sodium azide 1g/l, Tween 80 200 μ l/l.With regard to solidifying
The content of enzyme in hemase solution, in tt reagent, the content of enzyme is 4ku/l；In fib reagent, the content of enzyme is 70ku/l.
Embodiment 10
The content range of hepes acid in thrombin solution
According to table 11, with 15% as nature controlling line, in thrombin solution hepes acid content range can be 5.9g/l extremely
23.8g/l.
The content range of hepes acid in table 11 thrombin solution
Embodiment 11
The content range of calcium lactate in thrombin solution
According to table 12, with 15% as nature controlling line, in thrombin solution, the content range of calcium lactate can be 2g/l to 10g/l.
The content range of calcium lactate in table 12 thrombin solution
Embodiment 12
The content range of thrombin solution Glutamic Acid sodium
According to table 13, with 15% as nature controlling line, the content range of thrombin solution Glutamic Acid sodium can be 10g/l extremely
100g/l.
The content range of table 13 thrombin solution Glutamic Acid sodium
Embodiment 13
The content range of sodium azide in thrombin solution
According to table 14, with 15% as nature controlling line, in thrombin solution the content range of sodium azide can be 0.4g/l extremely
1.3g/l.
The content range of sodium azide in table 14 thrombin solution
Embodiment 14
The content range of Tween 80 in thrombin solution
According to table 15, with 15% as nature controlling line, in thrombin solution the content range of Tween 80 can be 100 μ l/l extremely
400μl/l.
The content range of Tween 80 in table 15 thrombin solution
Claims (10)
1. a kind of stabilizer for thrombin solution, it comprises water-soluble lactic acid compound and water solublity glutamic acid compounds.
2. stabilizer according to claim 1, wherein said water-soluble lactic acid compound and described water solublity glutamic acid compounds
Ratio be 1:50 to 50:1.
3. the stabilizer according to claim 1 or 2, wherein said water-soluble lactic acid compound is water-soluble lactic acid salt.
4. the stabilizer according to claim 1 or 2, wherein said water-soluble lactic acid compound is calcium lactate, EINECS 212-761-8, magnesium lactate
Or one of zinc lactate or two or more combinations.
5. the stabilizer according to claim 1 or 2, wherein said water solublity glutamic acid compounds are water-soluble glutamate.
6. stabilizer according to claim 4, wherein said water-soluble glutamate is sodium glutamate, Kaglutam or its group
Close.
7. the stabilizer according to any one of claim 1 to 6 is used for improving the purposes of thrombin solution stability.
8. the stabilizer according to any one of claim 1 to 6 is used for the purposes of reagent preparation.
9. purposes according to claim 8, described reagent is the reagent for detection fibers proteinogen or thrombin time.
10. a kind of method preparing thrombin solution, including the step using the stabilizer according to any one of claim 1 to 6
Suddenly.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107561296A (en) * | 2017-10-13 | 2018-01-09 | 山东艾科达生物科技有限公司 | One kind measure thrombin time（TT）Kit |
| CN108195783A (en) * | 2018-01-30 | 2018-06-22 | 迈克生物股份有限公司 | Heparin activity determination kit |
| CN113005176A (en) * | 2019-12-20 | 2021-06-22 | 深圳市帝迈生物技术有限公司 | Stabilizer, prothrombin time detection reagent, preparation method and kit thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107561296A (en) * | 2017-10-13 | 2018-01-09 | 山东艾科达生物科技有限公司 | One kind measure thrombin time（TT）Kit |
| CN108195783A (en) * | 2018-01-30 | 2018-06-22 | 迈克生物股份有限公司 | Heparin activity determination kit |
| CN113005176A (en) * | 2019-12-20 | 2021-06-22 | 深圳市帝迈生物技术有限公司 | Stabilizer, prothrombin time detection reagent, preparation method and kit thereof |
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