CN106337044B - Thrombin solution - Google Patents
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- CN106337044B CN106337044B CN201610874955.9A CN201610874955A CN106337044B CN 106337044 B CN106337044 B CN 106337044B CN 201610874955 A CN201610874955 A CN 201610874955A CN 106337044 B CN106337044 B CN 106337044B
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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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- C12N9/64—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 the stabilizer of thrombin solution, thrombin solution and relevant detection reagent, prepare the method for stable thrombin solution and the purposes of the stabilizer of thrombin solution.
Description
Technical field
The present invention relates to enzyme industrial circle and field of immunodetection, relate in particular to thrombin solution stabilizer,
Thrombin solution and relevant detection reagent 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 generated by hepatic parenchymal cells.About 80% of fibrinogen in organism is distributed in blood plasma (in normal adult about
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 chains form fibrin by the way that fibrin ferment is decomposed, and are generated to play thrombus, hemostasis blood
The main function of bolt.Clinically, increase in inflammation, reduced in hepatosis, DIC of height etc..
The clinical meaning of fibrinogen assay includes but not limited to：1. pathologic increases：(1) prethrombotic state and thrombus
Property disease when, body coagulation function enhancing, plasma fibrinogen increases, such as acute myocardial infarction, diabetes, the hypertension of pregnancy
Disease, atherosclerosis, malignant tumour etc..(2) albumen synthesis increases, such as connective tissue disease, Huppert's disease.(3) anti-
Answering property increases, such as after acute infection, acute nephritis, burn, shock, major operation.2. pathologic reduces：(1) consumption is excessive, leads
Plasma content is caused to reduce, such as DIC.(2) Fibrinolytic Activities enhance, and FIB is decomposed, such as primary hyperfibrinolysis disease.(3)
Synthesis is reduced, such as serious hepatitis, hepatic sclerosis.
The measuring method of fibrinogen includes fibrin ferment additive process (Clauss methods), Clotting-time method (PT algorithms), exempts from
Epidemic disease turbidimetry (TIA), emulsion technique etc. generally use fibrin ferment additive process.
Under fibrin ferment additive process, the content of fibrinogen ultimately forms fibre according to fibrinogen and thrombin action
The principle of fibrillarin measures, and standard curve is made by reference blood plasma of international standard substance, with fibrin ferment come when measuring the clotting of plasma
Between, i.e., thrombin time (Thrombin Time, TT), gained plasma coagulation time are in negative with fibrinogen concentration in blood plasma
Correlation, to obtain the content of fibrinogen.
Specifically, thrombin time refers to the time of the clotting of plasma after standardized factor is added in blood plasma.
Under thrombin action, the fibrinogen in blood plasma to be checked is changed into fibrin.When anticoagulant substances in blood plasma to be checked
When increasing, thrombin time extends.When blood plasma to be checked is in situations such as acid, test object is with abnormal fibrous proteinemia
Under, thrombin time shortens.
For example, thrombin time extension sees plasma fibrinogen attenuating or textural anomaly；Clinical application heparin, or
Heparin sample anticoagulant substances when hepatopathy, nephrosis and systemic loupus erythematosus increase；Fibrinolytic system function is hyperfunction.
The fibrin ferment used in fibrinogen assay, cuts peptide from the factor as its precursor and forms tool
There is α-fibrin ferment of coagulation activity.Known this α-fibrin ferment meeting selfdecomposition is solidifying at the β-without coagulation activity of low molecular weight
Hemase, and then resolve into γ-fibrin ferment when long-term preservation, is usually freeze-dried.
For measuring the reagent of fibrinogen (FIB) content or thrombin time (TT), existing reagent is
Freeze-dried reagent in use, is required to redissolve in addition to expensive, inconvenient to use, and will necessarily bring and redissolve
Volumetric errors are caused in journey, and result is caused deviation occur.And this problem is not present in liquid reagent, and it is easy to use, that is, it opens
It uses, difference between batch smaller is as a result more acurrate.Therefore urgently need to develop the stabilizer that can stablize thrombin solution and
Stable thrombin solution product.
Invention content
It is molten to improve fibrin ferment for the defect that the technical problem to be solved by the present invention is to be not easy to preserve for thrombin solution
The stability of liquid reduces to extend the holding time and preserves cost, easy to use, reduces experimental error.
Present inventors discovered unexpectedly that gluconic acid radical ion is molten with fibrin ferment is stablized with glutamate ion
The synergistic effect of liquid.
The present invention provides the stabilizers for thrombin solution, and it includes Water-Soluble Glucose acid compounds and water solubility
Glutamic acid compounds.In one embodiment, the stabilizer is by Water-Soluble Glucose acid compound and water-soluble glutamic acid
Compound forms.In another embodiment, the stabilizer includes Water-Soluble Glucose acid compound, water-soluble glutamic acid
Compound and solvent.In another embodiment in addition, the stabilizer includes Water-Soluble Glucose acid compound, water
Dissolubility glutamic acid compounds, solvent and those skilled in the art being capable of conventional use of auxiliary element (such as buffer, surfaces
Activating agent, preservative etc.).The solvent, which can be water, organic solvent or those skilled in the art, routinely to be determined or make
The combination of one or more of solvent.Preferably, the solvent is water.
Preferably, in the stabilizer for thrombin solution of the invention, Water-Soluble Glucose acid compound and water solubility
The ratio of glutamic acid compounds is 1:50 to 50:1.Preferably, the upper limit of the ratio can be 40:1、30:1、20:1、10:
1、9:1、8:1、7:1、6:1、5:1、4:1、3:1、2:1, the lower limit of the ratio can be 1:40、1:30、1:20、1:10、1:
9、1:8、1:7、1:6、1:5、1:4、1:3、1:2, the ratio can be above-mentioned upper limit value and the range that lower limiting value arbitrarily combines.
It is highly preferred that the ratio can be 1:1.
Preferably, the Water-Soluble Glucose acid compound can be Water-Soluble Glucose hydrochlorate.The water solubility grape
Sugar lime can be the combination of one or more of calcium gluconate, zinc gluconate or magnesium gluconate.Preferably,
The Water-Soluble Glucose hydrochlorate is calcium gluconate or zinc gluconate.
Water-Soluble Glucose acid compound used in the present invention is commercially available, such as from Shanghai Aladdin biochemistry section
The trade mark of skill limited liability company (Shanghai Jing Chun biochemical technologies limited liability company) is the gluconic acid of Aladdin (Aladdin)
Calcium (for example, article No. C110858), the zinc gluconate etc. from lark prestige Science and Technology Ltd..
Preferably, the water-soluble glutamic acid compounds can be water-soluble glutamate.It is highly preferred that the water solubility
Glutamate is sodium glutamate, potassium glutamate or combinations thereof.Most preferably, the water-soluble glutamate is sodium glutamate.
Water solubility glutamic acid compounds used in the present invention are commercially available, such as are had from Shanghai sky biological reagent
The sodium glutamate of limit company, the trade mark from the Shanghai bio tech ltd Yuan Ye are the potassium glutamate of source leaf (for example, article No.
S20427) etc..
The present invention also provides the thrombin solutions of the stabilizer comprising the present invention.
The thrombin solution that the stabilizer of the present invention is applicable in is (referred to as " thrombin solution of the invention ") to make fibrin ferment
The mixing of aqueous solution, organic solvent solution or water and organic solvent made of being suspended or dissolved in water and/or organic solvent
Solution, preferably aqueous solution or water and organic solvent mixed solution.The organic solvent, as long as point of fibrin ferment will not be caused
Solution and activity suppression etc., and its final use is not influenced, there is no particular limitation, such as dimethyl sulfoxide (DMSO), glycerine, poly- second
Glycol, polypropylene glycol etc..One or more of these substances can also be applied in combination.
Preferably, it in thrombin solution of the invention, is calculated with the total volume of thrombin solution, Water-Soluble Glucose acidification
The concentration for closing object is 1g/L to 50g/L；It is highly preferred that in the thrombin solution of the present invention, with the total volume meter of thrombin solution
It calculates, the concentration of Water-Soluble Glucose acid compound is 2g/L to 40g/L；It is highly preferred that in the thrombin solution of the present invention, with solidifying
The total volume of hemase solution calculates, and the concentration of Water-Soluble Glucose acid compound is 3g/L to 30g/L；It is highly preferred that of the invention
Thrombin solution in, calculated with the total volume of thrombin solution, the concentration of Water-Soluble Glucose acid compound be 3g/L extremely
25g/L；It is highly preferred that in the thrombin solution of the present invention, calculated with the total volume of thrombin solution, Water-Soluble Glucose acidification
Close object concentration be 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, 15g/L, 20g/L, 25g/L,
30g/L.It is further preferred that in the thrombin solution of the present invention, calculated with the total volume of thrombin solution, Water-Soluble Glucose
The concentration of acid compound is 7.7g/L.
Preferably, it in thrombin solution of the invention, is calculated with the total volume of thrombin solution, water-soluble glutamic acid chemical combination
The concentration of object 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
It calculates, the concentration of water-soluble 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 total volume of hemase solution calculates, the concentration of water-soluble 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, calculated with the total volume of thrombin solution, water-soluble paddy
The concentration of propylhomoserin compound is 50g/L.
The thrombin solution of the present invention can also include buffer, surfactant and preservative.
Preferably, the buffer can be 4- hydroxyethyl piperazineethanesulfonic acids (N- (2- ethoxys) piperazine-N'-2- ethane
Sulfonic acid；Molecular formula:C8H18N2O4S, also known as HEPES acid), citric acid, phosphoric acid, acetic acid, imidazoles, 3- (N- morpholines) third sulphur
(MOPS), two (2- ethoxys) imido grpup three (methylol) methane (BIS-TRIS), trishydroxymethylaminomethane (TRIS), 3-
(N- morpholinyls) -2- hydroxy-propanesulfonic acids (MOPSO), N- (2- acetylaminos)-imino-diacetic acetic acid (ADA) or 2-morpholine ethane sulfonic acid
One or more of (MES) combination；It is highly preferred that buffer is 4- hydroxyethyl piperazineethanesulfonic acids.
Buffer used in the present invention is commercially available, such as the 4- from Suzhou City Bake bio tech ltd
Hydroxyethyl piperazineethanesulfonic acid, 3- (N- morpholinyls) -2- hydroxyls third from uncommon love (Shanghai) the chemical conversion industry Development Co., Ltd of ladder
The 2- morpholines that sulfonic acid (for example, product coding H0671), the trade mark from Sa En chemical technologies (Shanghai) Co., Ltd. are An Naiji
Ethanesulfonic acid (for example, goods number D090002).
There is no particular limitation as long as the amount with buffer capacity for the additive amount of the buffer, people in the art
Member can routinely determine the content for the buffer for being suitble to use.But the inventors found that the fibrin ferment of the present invention is molten
It in liquid, is calculated with the total volume of thrombin solution, when the concentration of buffer is following values range, is more advantageous to and realizes this hair
Bright purpose：Preferably, it in thrombin solution of the invention, is calculated with the total volume of thrombin solution, the concentration of buffer is
1g/L to 50g/L；It is highly preferred that in the thrombin solution of the present invention, calculated with the total volume of thrombin solution, buffer it is dense
Degree is 5g/L to 24g/L；It is highly preferred that in the thrombin solution of the present invention, calculated with the total volume of thrombin solution, buffer
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 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, calculated with the total volume of thrombin solution, the concentration of buffer is
11.9g/L。
The surfactant can be anionic surfactant, cationic surfactant, amphoteric ion table
The combination of one or more of face activating agent or nonionic surfactant.
The anionic surfactant can be lauryl sodium sulfate, dodecyl sodium sulfate, dodecyl-N-
The combination of one or more of sodium sarcosinate, sodium taurocholate, NaTDC or sodium taurodeoxycholate.
The cationic surfactant can be cetyl trimethylammonium bromide, four decyl ammonium bromides or dodecane
The combination of one or more of pyridinum chloride.
The zwitterionic surfactant can be 3- [(3- courages amido propyl) dimethyl ammonium] -1- propane sulfonic acid salt, 3-
[(3- courages amido propyl) dimethyl ammonium] -2- hydroxyl -1- propane sulfonic acid salt, palmityl lysolecithin, dodecyl-N- glycine betaines
Or the combination of one or more of dodecyl-Beta-alanine.
The nonionic surfactant can be octyl glucoside, heptyl glucosinolate, capryl-N- methyl Portugal
Osamine, polyoxyethylene lauryl ether, seven methylhexyl ether of polyoxyethylene, Triton X100, polyoxyethylene nonyl
One or both of base phenyl ether, polyoxyethylene fatty acid ester, sucrose fatty ester or polyoxyethylene sorbitol ester with
On combination.
Preferably, the surfactant is nonionic surfactant；It is highly preferred that the surfactant is poly-
Ethylene oxide Sorbitan Monooleate (also known as Tween 80).
Surfactant used in the present invention is commercially available, such as the tween from Chengdu Ke Long chemical reagents factory
80, come from Shanghai Aladdin biochemical technology limited liability company (Shanghai Jing Chun biochemical technologies limited liability company) trade mark be Ah
The Tween 80 (for example, article No. T104866) of Latin (Aladdin), the trade mark of the Shanghai bio tech ltd Yuan Ye are source leaf
Sucrose fatty ester (for example, article No. S30894) etc..
As long as the additive amount of the surfactant makes the stability-enhanced amount of thrombin solution, without special
It limits, those skilled in the art can routinely determine the content for the surfactant for being suitble to use.But the present inventor
It was found that in the thrombin solution of the present invention, calculated with the total volume of thrombin solution, when the concentration of surfactant is following number
When being worth range, it is more advantageous to and achieves the object of the present invention：Preferably, it is calculated with the total 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 total volume of thrombin solution, the concentration of surfactant is
50 μ l/L to 500 μ l/L；It is highly preferred that calculated with the total 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 total 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 total volume of thrombin solution, the concentration of surfactant
It is 200 μ l/L.
The preservative can be Sodium azide (NaN3), Ciprofloxacin, one or both of propionic acid or sodium benzoate with
On combination.Preferably, the preservative is Sodium azide.
Preservative used in the present invention is commercially available, such as comes precious biotechnology (Shanghai) limited liability company westerly
Trade mark be Amresco Sodium azide (for example, article No. be 0639), come from Chengdu Hua Xia chemical reagent Co., Ltd Sodium azide
(the brilliant pure biochemical technology share in Shanghai is limited for (for example, article No. is H1100046), Shanghai Aladdin biochemical technology limited liability company
Company) trade mark be Aladdin (Aladdin) sodium benzoate (for example, article No. is S104128), come from Sigma-Aldrich
The preservative that the trade mark of Co., Ltd (Sigma-Aldrich LLC) is Proclin300 is (for example, product identification is
48914-U) etc..
There is no particular limitation as long as in prescribed limit for the additive amount of the preservative.Those skilled in the art can be with
The conventional content for determining the preservative for being suitble to use.But the inventors found that the present invention thrombin solution in, with
The total volume of thrombin solution calculates, and when the concentration of preservative is following values range, is more advantageous to the mesh for realizing the present invention
's：Preferably, it is calculated with the total volume of thrombin solution, the concentration of preservative is 0.1g/L to 5.0g/L；It is highly preferred that with solidifying
The total volume of hemase solution calculates, and the concentration of preservative is 0.2g/L to 2.5g/L；It is highly preferred that with the totality of thrombin solution
Product calculates, and the concentration of preservative is 0.4g/L to 1.3g/L；It is further preferred that being calculated with the total 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 total volume of thrombin solution, the concentration of preservative is 1.0g/L.
The fibrin ferment used in the present invention can be the fibrin ferment of the animal origins such as people, pig, ox, by genetic engineering legal system
Standby fibrin ferment or commercially available drug.Fibrin ferment used in the present invention is commercially available, e.g. comes from one lattice pharmacy of Hunan
The fibrin ferment freeze-dried powder of Co., Ltd, coagulating from Sigma-Aldrich Co., Ltd (Sigma-Aldrich LLC)
Hemase preparation (for example, product identification is 10602400001) etc..
The additive amount of fibrin ferment in the thrombin solution of the present invention is as long as being suitble to the purpose of the present invention without special
It limits.It was found by the inventors of the present invention that in the thrombin solution of the present invention, calculated with the total volume of thrombin solution, fibrin ferment
Concentration can be 1KU/L to 120KU/L；It is highly preferred that in the thrombin solution of the present invention, with the total volume of thrombin solution
Calculate, the concentration of fibrin ferment 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 total volume of thrombin solution, fibrin ferment it is 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 are 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 total volume of enzyme solutions calculates, and the concentration of fibrin ferment can be 2KU/L, 4KU/L, 30KU/L, 70KU/L, 120KU/L.
Preferably, thrombin solution of the invention is calculated with the total volume of thrombin solution, including concentration is 7.7g/L
Calcium gluconate, concentration are that be 4- hydroxyethyl piperazineethanesulfonic acids, the concentration of 11.9g/L be for the sodium glutamate of 50g/L, concentration
The Sodium azide of 1.0g/L, concentration are the polyoxyethylene sorbitan monooleates of 200 μ l/L.It is highly preferred that above-mentioned fibrin ferment
In solution, including concentration is the fibrin ferment of 2KU/L, 4KU/L, 30KU/L or 70KU/L.
Preferably, thrombin solution of the invention is calculated with the total volume of thrombin solution, including concentration is 7.7g/L
Zinc gluconate, concentration are that be 4- hydroxyethyl piperazineethanesulfonic acids, the concentration of 11.9g/L be for the sodium glutamate of 50g/L, concentration
The Sodium azide of 1.0g/L, concentration are the polyoxyethylene sorbitan monooleates of 200 μ l/L.It is highly preferred that above-mentioned fibrin ferment
In solution, including concentration is the fibrin ferment of 2KU/L, 4KU/L, 30KU/L or 70KU/L.
The present invention also provides the methods for preparing thrombin solution, and the method includes using the step of the stabilizer of the present invention
Suddenly.
The present invention also provides the purposes that the stabilizer of the present invention is used to improve thrombin solution stability.
The present invention also provides the purposes that the thrombin solution of the stabilizer of the present invention or the present invention is used to prepare reagent.
The present invention also provides a kind of reagents, contain the stabilizer of the present invention or the thrombin solution of the present invention.
The reagent prepared using the stabilizer of the present invention or the thrombin solution of the present invention or the stabilization containing 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, the reagent is the reagent for detecting fibrinogen or thrombin time.
Preferably for the present invention for detecting the reagent of fibrinogen, fibrin ferment in thrombin solution contains
Amount is 30KU/L or 70KU/L；For the present invention for detecting the reagent of thrombin time, the fibrin ferment in thrombin solution
Content be 2KU/L or 4KU/L.
The present invention also provides a kind of kits, and it includes the reagents of the present invention.
The present inventors have noted that in the case where thrombin solution other components are constant, under different concentration of thrombin, contain
There is the measured value of the reagent of the thrombin solution of the present invention that can integrally improve, but this thrombin solution and correlation to the present invention
The implementation of the stability of product itself and relevant purposes, method does not have an impact, and the raising of measured value will not interfere this hair
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 realize above-mentioned target with the content of general adjustment fibrin ferment.
The thrombin solution of stabilizer containing the present invention is easy to use, without redissolving, avoids due to redissolving volume difference
Lead to error.Plug and play, difference between batch smaller are as a result more acurrate.
The thrombin solution stability prepared using the stabilizer of the present invention is strong, and 2-8 DEG C is stablized 1 year or more, 37 DEG C of stabilizations
20 days or more, 2-8 DEG C of corkage stability 10 days or more.Stabilizer of the invention can improve the stabilization of thrombin solution as a result,
Property, improve the storage form and condition of Related product, reduces the cost of production and application.
Specific implementation mode
The each component source used in the following embodiment of the present invention is respectively：From Shanghai Aladdin biochemical technology share
The trade mark of Co., Ltd (Shanghai Jing Chun biochemical technologies limited liability company) is the calcium gluconate (example of Aladdin (Aladdin)
Such as, article No. C110858), the zinc gluconate from lark prestige Science and Technology Ltd., come from the limited public affairs of Shanghai source leaf biotechnology
The trade mark of department be the potassium glutamate (for example, article No. S20427) of source leaf, Shanghai sky biological reagent Co., Ltd sodium glutamate,
The 4- hydroxyethyl piperazineethanesulfonic acids (HEPES acid) of Suzhou City Bake bio tech ltd, Chengdu Ke Long chemical reagents factory
Tween 80, precious biotechnology (Shanghai) limited liability company in west trade mark be the Sodium azide (article No. 0639) of Amresco, Hunan one
The fibrin ferment freeze-dried powder of lattice pharmaceutical Co. Ltd.
Embodiment 1
The preparation of thrombin time (TT) detection reagent of the stabilizer containing the present invention
HEPES acid 11.9g, calcium gluconate 7.7g, sodium glutamate 50g, Sodium azide 1g, Tween 80 0.2g are weighed, is added
It is dissolved in distilled water, adjusts pH to 6.4 with sodium hydroxide, be settled to 1L.Fibrin ferment 2 (2000U/ branch) is taken, is prepared with above
Buffer solution 1mL/ branch dissolves, and takes dissolving fibrin ferment 3.6mL (adjustable enzyme amount control difference between batch), is added in 1L buffer solutions and shakes up,
TT detection reagents, which are prepared, to be completed.
Product performance index
1. repeatability：The coefficient of variation (CV)≤5%；With normal Quality Control blood plasma retest 10 times, being averaged for measurement is calculated
ValueWith standard deviation (s).The coefficient of variation (CV) is calculated according to formula (1).Acquired results should meet wanting for following 3. stability
It asks.
In formula：
In S：Standard deviation
--- measure mean value
2. difference between batch：The coefficient of variation (CV)≤10%；Test the reagent of 3 different batches respectively with normal Quality Control blood plasma,
Each batch measures retest 10 times, calculates 30 measured value average valuesWith standard deviation (s), calculated according to formula (1)
The coefficient of variation (CV).
3. stability
2 DEG C~8 DEG C closed preservations of this product are newly opened one bottle of measurement Quality Control blood plasma, were compared with first day, relative deviation exists every time
In 15%, the term of validity 12 months.
Embodiment 2
The detection method of thrombin time (TT) detection reagent
100 μ L of blood plasma are taken, 37 DEG C preheat 2 minutes, 100 μ L of thrombin reagent are then added, in CA550 full automatic blood-coagulation instrument
(producer SYSMEX) is measured according to the specification of producer.
Points for attention：
1. blood sampling should avoid haemolysis, tissue fluid is prevented to be mixed into.Smooth when blood drawing, anti-freezing is abundant, can not there is sludged blood；
Blood platelet must be removed when separated plasma.
2. when sample collection anti-coagulants should not be made with EDTA and heparin.The ratio of blood and anti-coagulants should be accurate, blood sampling volume
Sample of the deviation more than 10% should not use.
3. if hematocrit≤20% or >=55%, need the ratio of adjustment blood sample and anti-coagulants, anti-coagulants ml
=0.00185 × blood ml × (100- hematocrits).
Measuring pipette and sample injector after 4. use is calibrated.
5. using the plastics of cleanliness without any pollution or the glassware of silication.
6. experimental temperature is controlled at 37 DEG C ± 1.0 DEG C.
7. reagent can change in proportion as needed with sample size.
8. reagent use finishes, bottleneck should be wiped clean, screws bottle cap as early as possible, puts back under the condition of storage of requirement and protects in time
It deposits.
Embodiment 3
The preparation of fibrinogen (FIB) detection reagent of the stabilizer containing the present invention
(1) reagent preparation：HEPES acid 11.9g, calcium gluconate 7.7g, sodium glutamate 50g are weighed, Sodium azide 1g is spat
80 0.2g of temperature are added in distilled water and dissolve, and adjust pH to 6.4 with sodium hydroxide, are settled to 1L.Take 30 (3000U/ of fibrin ferment
Branch), the dissolving of buffer solution 1mL/ branch is prepared with above, is all added in 1L buffer solutions and shakes up, FIB detection reagents, which are prepared, to be completed.
(2) prepared and diluted sample buffer：Weigh imidazoles 3.06g, sodium chloride 5.22g, sodium citrate 1.2g, Sodium azide
0.95g is added in distilled water and dissolves, and adjusts pH to 7.30 with sodium hydroxide, is settled to 1L.After stable in two days, pH is adjusted again
To 7.30.
Product performance index
1. accuracy：Reference material or correctness Quality Control object is taken to test, in triplicate, average value is obtained, according to formula (2)
Average value and reference material or the relative deviation of main calibration object are calculated, as a result should meet relative deviation should be in ± 15% range
It is required that.Calculation formula：
(calculation formula：Substance or main calibration object relatively partially
Difference ... ... ... (2)
In formula：
X-- detects mean value；
Xc-- reference materials or correctness Quality Control object sign value.
2. repeatability
Distinguish retest 10 times with normal Quality Control blood plasma and abnormal Quality Control blood plasma, and calculates the average value of measurementWith
Standard deviation (S).The coefficient of variation (CV) is calculated by formula (1), as a result should meet the requirement of the coefficient of variation (CV)≤8%.
3. difference between batch
The reagent of 3 different batches is tested with normal Quality Control blood plasma, each batch is tested 10 times, and it is flat to calculate 30 measured values
Mean valueWith standard deviation (S), the coefficient of variation (CV) is calculated according to formula (1), as a result should meet the coefficient of variation (CV)≤15%
Requirement.
4. stability
One bottle of measurement Quality Control blood plasma is newly opened in 2 DEG C~8 DEG C closed preservations of this product every time, and relative deviation is in 15%, the term of validity
12 months.
Embodiment 4
The detection method of fibrinogen (FIB) detection reagent
(1) standard curve is established
Reference blood plasma is diluted with dilution according to the form below, you can obtains series concentration：
FIB detection reagents pre-temperature is taken 3 minutes, until 37 DEG C.Take the reference blood plasma 100 μ l after dilution, 37 DEG C of pre-temperatures 2 minutes.
50 μ l pre-temperature reagents are added, at once mixing and timing, record setting time.It is with the obtained target level of reference blood plasma gradient dilution
Abscissa, corresponding setting time (second) is ordinate, 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 carries out 10 times of dilutions, preparation of the determination step with working curve with dilution buffer.It is automatically coagulated with CA550
Blood instrument (producer SYSMEX) is measured according to the specification of producer.
Points for attention：
1. blood sampling should avoid haemolysis, tissue fluid is prevented to be mixed into.Smooth when blood drawing, anti-freezing is abundant, can not there is sludged blood；
Blood platelet must be removed when separated plasma.
2. when sample collection anti-coagulants should not be made with EDTA and heparin.The ratio of blood and anti-coagulants should be accurate, blood sampling volume
Sample of the deviation more than 10% should not use.
3. if hematocrit≤20% or >=55%, need the ratio of adjustment blood sample and anti-coagulants, anti-coagulants ml
=0.00185 × blood ml × (100- hematocrits).
Measuring pipette and sample injector after 4. use is calibrated.
5. using the plastics of cleanliness without any pollution or the glassware of silication.
6. experimental temperature is controlled at 37 DEG C ± 1.0 DEG C.
7. reagent can change in proportion as needed with sample size.
8. reagent use finishes, bottleneck should be wiped clean, screws bottle cap as early as possible, puts back under the condition of storage of requirement and protects in time
It deposits.
Embodiment 5
Exist between Water-Soluble Glucose acid compound and water-soluble glutamic acid compounds and stablizes cooperateing with for thrombin solution
Effect
Under conditions of its dependent variable is consistent, for only containing sodium glutamate containing calcium gluconate, only, containing Portugal simultaneously
Grape Calciofon and sodium glutamate and four kinds of thrombin solutions simultaneously containing calcium gluconate and potassium glutamate measure at 37 DEG C
Stabilization time, be nature controlling line with relative deviation 10%, observe the testing result of above-mentioned thrombin solution.Testing result takes 5 times
The mean value of experiment.
The content of each ingredient of thrombin solution is respectively in table 1：Calcium gluconate 7.7g/L (such as containing), sodium glutamate
50g/L (such as containing), potassium glutamate 50g/L (such as containing), HEPES acid 11.9g/L, Sodium azide 1g/L, 200 μ l/L of Tween 80,
Fibrin ferment 2KU/L.
Table 1：Calcium gluconate stablizes the synergistic effect of thrombin solution with water-soluble glutamic acid compounds
2 calcium gluconate of table stablizes the experiment of thrombin solution with water-soluble glutamic acid compounds
From table 1, table 2 it is observed that when calcium gluconate is existed simultaneously with water-soluble glutamic acid compounds, fibrin ferment is molten
The stabilization number of days longest of liquid.Therefore, exist between calcium gluconate and water-soluble glutamic acid compounds and stablize thrombin solution
Synergistic effect.
The present invention also has chosen the thrombin solution containing zinc gluconate and sodium glutamate further to verify water solubility
There is the synergistic effect for stablizing thrombin solution between gluconic acid compound and water-soluble glutamic acid compounds.It measures at 37 DEG C
Stabilization time, be nature controlling line with relative deviation 10%, observe the testing result of above-mentioned thrombin solution.Testing result takes 3 times
The mean value of experiment.
Above-mentioned zinc gluconate and sodium glutamate contain as stabilizer as each ingredient in the thrombin solution of stabilizer
Amount is respectively：Zinc gluconate 7.7g/L, sodium glutamate 50g/L, HEPES acid 11.9g/L, Sodium azide 1g/L, 200 μ of Tween 80
L/L, fibrin ferment 30KU/L (FIB reagents) or 2KU/L (TT reagents).The stability test of above-mentioned thrombin solution is as follows.
3 zinc gluconate of table stablizes the experiment of thrombin solution with sodium glutamate
Also there is good synergisticing stable effect by 3 visible zinc gluconate of table and sodium glutamate.
Therefore, it can be seen that between Water-Soluble Glucose acid compound and water-soluble glutamic acid compounds from above-mentioned experiment
In the presence of the synergistic effect for stablizing thrombin solution.
Embodiment 6
Stablizing effect of the amino acid or its salt of calcium gluconate and other non-glutamic acids to thrombin solution
The content of each ingredient is respectively in thrombin solution：Calcium gluconate 7.7g/L, glycine 50g/L (such as containing),
Arginine 50g/L (such as containing), it serine 50g/L (such as containing), glutamic acid 50g/L (such as containing), HEPES acid 11.9g/L, folds
Nitrogen sodium 1g/L, 200 μ l/L of Tween 80.Fibrin ferment is 2KU/L.
It is nature controlling line with relative deviation 10%, according to the experimental result of table 4,5 in table 1 in embodiment 5 and the present embodiment,
Calcium gluconate can not realize that water-soluble glutamic acid compounds are molten to fibrin ferment with the amino acid of other non-glutamic acids or its salt
The stablizing effect of liquid.
Table 4：Calcium gluconate stablizes the synergistic effect of thrombin solution with the amino acid of other non-glutamic acids
Table 5：Calcium gluconate stablizes the synergistic effect of thrombin solution with glycine or valine
In embodiment 7-11, in thrombin solution when a certain component content variation, the content of other compositions follows following number
Value：Calcium gluconate 7.7g/L, sodium glutamate 50g/L, HEPES acid 11.9g/L, Sodium azide 1g/L, 200 μ l/L of Tween 80.It closes
The content of enzyme in thrombin solution, in TT reagents, the content of enzyme is 2KU/L；In FIB reagents, the content of enzyme is 30KU/L.
Embodiment 7
The content range of HEPES acid in thrombin solution
Be nature controlling line with 15% according to table 6, in thrombin solution the content range of HEPES acid can be 5.9g/L extremely
23.8g/L。
The content range of HEPES acid in 6 thrombin solution of table
Embodiment 8
The content range of calcium gluconate in thrombin solution
Be nature controlling line with 15% according to table 7, in thrombin solution the content range of calcium gluconate can be 3g/L extremely
25g/L。
The content range of calcium gluconate in 7 thrombin solution of table
Embodiment 9
The content range of thrombin solution Glutamic Acid sodium
Be nature controlling line with 15% according to table 8, the content range of thrombin solution Glutamic Acid sodium can be 10g/L extremely
100g/L。
The content range of 8 thrombin solution Glutamic Acid sodium of table
Embodiment 10
The content range of Sodium azide in thrombin solution
Be nature controlling line with 15% according to table 9, 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 9 thrombin solution of table
Embodiment 11
The content range of Tween 80 in thrombin solution
Be nature controlling line with 15% according to table 10, 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 10 thrombin solution of table
Claims (10)
1. a kind of thrombin solution, it includes stabilizers, wherein the stabilizer includes Water-Soluble Glucose acid compound and water
Dissolubility glutamic acid compounds, buffer, surfactant and preservative, wherein the Water-Soluble Glucose acid compound is water-soluble
Property gluconate, the Water-Soluble Glucose hydrochlorate is one kind in calcium gluconate, zinc gluconate or magnesium gluconate
Or two or more combinations, and the water-soluble glutamic acid compounds are water-soluble glutamates, wherein with thrombin solution
Total volume calculates, and the concentration of Water-Soluble Glucose acid compound is 1g/L to 20g/L, and the concentration of water-soluble glutamic acid compounds is
1g/L to 150g/L, the buffer are 4- hydroxyethyl piperazineethanesulfonic acids, citric acid, phosphoric acid, acetic acid, imidazoles, 3- (N- morphines
Quinoline) the third sulphur, two (2- ethoxys) imido grpup three (methylol) methane, trishydroxymethylaminomethane, 3- (N- morpholinyls) -2- hydroxyls
The combination of one or more of propane sulfonic acid, N- (2- acetylaminos)-imino-diacetic acetic acid or 2-morpholine ethane sulfonic acid, it is described
Surfactant is anionic surfactant, cationic surfactant, zwitterionic surfactant or nonionic
The combination of one or more of property surfactant, the preservative is Sodium azide, Ciprofloxacin, propionic acid or benzoic acid
The combination of one or more of sodium.
2. thrombin solution according to claim 1, wherein the Water-Soluble Glucose acid compound and the water-soluble paddy ammonia
The ratio of acid compound is 1:50 to 50:1.
3. thrombin solution according to claim 1, wherein the water-soluble glutamate be sodium glutamate, potassium glutamate or its
Combination.
4. thrombin solution according to claim 1, wherein the anionic surfactant is lauryl sodium sulfate, ten
Dialkyl sulfonates, dodecyl-N- sodium sarcosinates, sodium taurocholate, NaTDC or one kind in sodium taurodeoxycholate or
Two or more combinations.
5. thrombin solution according to claim 1, wherein the cationic surfactant is cetyl trimethyl bromine
Change the combination of one or more of ammonium, four decyl ammonium bromides or dodecanyl pyridinium chloride.
6. thrombin solution according to claim 1, wherein the zwitterionic surfactant is 3- [(3- courages amido propyl)
Dimethyl ammonium] -1- propane sulfonic acid salt, 3- [(3- courages amido propyl) dimethyl ammonium] -2- hydroxyl -1- propane sulfonic acid salt, palmityl haemolysis
The combination of one or more of lecithin or dodecyl-N- glycine betaines or dodecyl-Beta-alanine.
7. thrombin solution according to claim 1, wherein the nonionic surfactant is octyl glucoside, heptyl sulphur
For glucoside, capryl-N-METHYL-ALPHA-L-GLUCOSAMINE, polyoxyethylene lauryl ether, seven methylhexyl ether of polyoxyethylene, polyoxyethylene
Isooctyl phenyl ether, ethylene nonyl phenyl ether, polyoxyethylene fatty acid ester, sucrose fatty ester or polyethylene oxide sorb
The combination of one or more of sugar alcohol ester.
8. a kind of reagent, including according to the thrombin solution of any one of claim 1 to 7.
9. reagent according to claim 8, for the reagent for detecting fibrinogen or thrombin time.
10. a kind of kit, it includes the reagents according to claim 8 or 9.
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|---|---|---|---|
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