CN104569446A - Liquid-type prothrombin time detection reagent and preparation method thereof - Google Patents
Liquid-type prothrombin time detection reagent and preparation method thereof Download PDFInfo
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- CN104569446A CN104569446A CN201510058508.1A CN201510058508A CN104569446A CN 104569446 A CN104569446 A CN 104569446A CN 201510058508 A CN201510058508 A CN 201510058508A CN 104569446 A CN104569446 A CN 104569446A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
Abstract
The invention discloses a liquid-type prothrombin time detection reagent and a preparation method thereof. The preparation method comprises the following steps: dissolving amino acid, polyethylene glycol, mannitol, bovine serum albumin, and gelatin in water so as to obtain a prothrombin protective fluid, and adjusting the pH value of the prothrombin protective fluid to 6.0-8.0; and dissolving prothrombin freeze-dried powder in the prothrombin protective fluid so as to obtain the liquid-type prothrombin time detection reagent, wherein the enzymatic activity of the liquid-type prothrombin time detection reagent is 5-80 IU/mL. The liquid-type prothrombin time detection reagent contains prothrombin, amino acid, polyethylene glycol, mannitol, bovine serum albumin and gelatin, and takes water as a solvent. The production process of the reagent disclosed by the invention is simplified, production facilities are simple, no freeze-drying process is performed, and the production cost is low; the reagent is used in an out-of-the-box mode; the variation among bottles is small; and the reagent is high in stability.
Description
Technical field
The present invention relates to external diagnosis reagent technical field, be specifically related to a kind of liquid-type clotting time (TT) and detect reagent and preparation method thereof.
Background technology
The principle that thrombin time (Thrombin time, TT) detects be appropriate fibrin ferment by the conversion of fibrinogen fibroblast cells in plasma sample, thus make the clotting of plasma, solidifying the required time is thrombin time (TT).
Thrombin time detects an easy test of blood coagulation, anti-freezing and fibrinolytic system function, and its detection has important value to clinical medical diagnosis on disease.
Thrombin time detection assay process is by the impact of many factors, wherein the quality of reagent is key factor, due to the problem of clotting reagent ubiquity unstable product quality domestic at present, susceptibility difference, cause testing result unstable, the result recorded between different Clinical Test Labs is difficult to compare, therefore the reagent overwhelming majority that clinical labororatory uses at present is imported product, expensive.On the other hand, due to important component in reagent---the stability of fibrin ferment is bad, and current thrombin reagent both domestic and external is almost freeze-dried powder formulation, needs to redissolve before using, and between troublesome poeration, bottle, difference is large, and after reagent redissolution, quality stability is poor.And although the reagent of liquid-type can directly use, handled easily, between bottle, difference is little, is more suitable for the operation of self-reacting device, because in long-term preservation process, stability is undesirable, limits its application.
Summary of the invention
This area needs the thrombin time developing a kind of stable liquid-type to detect reagent, based on this, the invention provides a kind of liquid thrombin time and detects reagent and preparation method thereof.
In order to realize foregoing invention object, this invention takes following technical scheme:
A kind of liquid thrombin time detects reagent, and containing fibrin ferment, amino acid, polyglycol, sweet mellow wine, bovine serum albumin(BSA), gelatin, and solvent is water.
Its pH value is 6.0 ~ 8.0.
Described amino acid is selected from glycocoll or alanine.
The molecular weight ranges of described polyglycol is at 8000-20000.
The enzymatic activity of described fibrin ferment is 5 ~ 80IU/mL.
Also comprise buffer salt.
Also comprise sodium azide.
Described amino acid whose quality percent by volume is 1% ~ 5%;
The quality percent by volume of polyglycol is 0.4% ~ 1%;
The quality percent by volume of sweet mellow wine is 3% ~ 10%;
The quality percent by volume of bovine serum albumin(BSA) is 3% ~ 12%;
The quality percent by volume of gelatin is 0.1% ~ 0.5%.
Liquid thrombin time detects a preparation method for reagent, comprising:
Amino acid, polyglycol, sweet mellow wine, bovine serum albumin(BSA), Gelatin are obtained in water fibrin ferment protection liquid, adjust ph 6.0 ~ 8.0;
Get fibrin ferment freeze-dried powder to be dissolved in described fibrin ferment protection liquid, obtain described liquid thrombin time and detect reagent, the enzymatic activity that described liquid thrombin time detects fibrin ferment in reagent is 5 ~ 80IU/mL.
Get fibrin ferment freeze-dried powder to be dissolved in described fibrin ferment protection liquid, the step obtaining described liquid thrombin time detection reagent comprises:
Get fibrin ferment freeze-dried powder be dissolved in described fibrin ferment protection liquid in obtain fibrin ferment mother liquor, in described fibrin ferment mother liquor, the enzymatic activity of fibrin ferment is 3000 ~ 10000IU/mL;
The described fibrin ferment protection liquid of described fibrin ferment mother liquor is diluted respectively, obtains some dilutability solution and titration, choose a dilutability according to titration results;
According to described dilutability, dilute described fibrin ferment mother liquor with described fibrin ferment protection liquid, obtain described liquid thrombin time and detect reagent.
Compared with prior art, the present invention has following beneficial effect:
1) reagent production run simplifies, and production equipment is simple, and without freeze-drying process, production cost is low;
2) using is model of instant use once to turn it on, does not need to redissolve, easy to operate;
3) there is not the difference that quality or volume difference because of water used cause, make a variation between bottle within 3%;
4) reagent stability is high, and between bottle, consistance is good, and 37 DEG C thermally-stabilised more than 30 days, 2 ~ 8 DEG C of sealed storage Absorbable organic halogens more than 18 months;
5) aggregative relative price, the powdered reagent that liquid reagent prepared by the inventive method generally uses compared with market has clear superiority.
Accompanying drawing explanation
Fig. 1 is that thrombin time of the present invention detects preparation method of reagent thereof schematic flow sheet.
Fig. 2 is that thrombin time of the present invention detects preparation method of reagent thereof flow process second schematic diagram.
Embodiment
The technological means realized to make the present invention, creation characteristic, reaching object and effect is easy to understand, setting forth the present invention further below in conjunction with concrete diagram.
With reference to Fig. 1, a kind of liquid thrombin time detects preparation method of reagent thereof, comprises the following steps:
Amino acid, polyglycol, sweet mellow wine, bovine serum albumin(BSA), Gelatin are obtained in water fibrin ferment protection liquid, adjust ph 6.0 ~ 8.0;
Get fibrin ferment freeze-dried powder to be dissolved in described fibrin ferment protection liquid, obtain described liquid thrombin time and detect reagent, the enzymatic activity that described liquid thrombin time detects fibrin ferment in reagent is 5 ~ 80IU/mL.
As shown in Figure 2, a kind of liquid thrombin time detects preparation method of reagent thereof, comprises the following steps:
Amino acid, polyglycol, sweet mellow wine, bovine serum albumin(BSA), Gelatin are obtained in water fibrin ferment protection liquid, adjust ph 6.0 ~ 8.0;
Get fibrin ferment freeze-dried powder be dissolved in described fibrin ferment protection liquid in obtain fibrin ferment mother liquor, in described fibrin ferment mother liquor, the enzymatic activity of fibrin ferment is 3000 ~ 10000IU/mL;
The described fibrin ferment protection liquid of described fibrin ferment mother liquor is diluted respectively, obtains some dilutability solution and titration, choose a dilutability according to titration results;
According to described dilutability, dilute described fibrin ferment mother liquor with described fibrin ferment protection liquid, obtain described liquid thrombin time and detect reagent.
Disclosed herein as well is a kind of liquid thrombin time and detect reagent, containing fibrin ferment, amino acid, polyglycol, sweet mellow wine, bovine serum albumin(BSA) and gelatin, solvent is water, and pH is 6.0-8.0.Wherein, the preferred glycocoll of amino acid or alanine, the polyglycol of polyglycol preferred molecular weight 8000-1000.
In the application, glycocoll, sweet mellow wine coordinate the stability that can keep fibrin ferment well in liquid reagent with other reagent component, the liquid-type reagent of preparation is 2 ~ 8 DEG C of sealed storage after 18 months, carry out thrombin time detection with this reagent, testing result skew is within 10%.In addition, this reagent can also contain buffer salt, to maintain the pH value of reagent better, to keep the activity of fibrin ferment better.This reagent can also contain antiseptic, with the growth of the thing that suppresses to support one's family in reagent, is conducive to the long-term preservation of this liquid-type reagent.The antiseptic of conventional diagnostic reagent all can use, such as Sodium azide.
Composition, the preparation method that a liquid thrombin time detects reagent is specifically introduced below with a specific embodiment, and performance for stability.
Embodiment 1 thrombin time detects the preparation method of reagent
1 fibrin ferment protection liquid preparation: take Tris6.06g (50mmol/L), glycocoll 12g (massfraction is 1.2%), PEG200005g (massfraction is 0.5%), BSA 35g (massfraction is 3.5%), gelatin 2g (massfraction is 0.2%), sweet mellow wine 40g (massfraction is 0.4%), Sodium azide 1g (massfraction is 0.1%); add 1L purified water; stirring makes it fully dissolve, and adjusts pH to be 7.0 with the NaOH of 0.1mol/L.
2 filtration sterilizations: with the Triafol T of water-wet 0.45 μm, are close to be placed on filtered off with suction device core and carry out suction filtration to protection liquid, collect the fibrin ferment protection liquid that filtrate obtains filtering, remove the insolubles in a small amount of preparation.
3 fibrin ferment mother liquor: the fibrin ferment dried frozen aquatic products getting 1 1mL specification, 50000IU/mL; adding 1mL fibrin ferment protection liquid, room temperature 10 minutes, turns upside down and makes it fully dissolve in interval; then be transferred in the small beaker that 9mL fibrin ferment protection liquid is housed, mixing obtains 10mL fibrin ferment mother liquor.
The dilution of 4 fibrin ferment mother liquors and titration:
1) with fibrin ferment protection liquid, by 1:100,1:150,1:200,1:400, dilution preparation thrombin time is carried out to fibrin ferment mother liquor and detect each 2mL of reagent, be designated as A, B, C, D tetra-groups respectively.
2) above-mentioned A, B, C, D tetra-groups of samples go up coagulo meter to 30 routine hospital reports respectively for fresh (in 2h) plasma sample and Siemens blood clotting normal level quality-control product Ci-Trol 1 carry out survey detection normally, detection method is: get test plasma or quality-control product 100 μ L, educate temperature 3 minutes for 37 DEG C, then add 100 μ L thrombin reagents, detect with Sysmex CA-1500 coagulo meter freezing method.
Detect and find that dilution ratio is within the scope of 1:150 ~ 1:400, each sample results, all in 20s, meets Industry code requirements.
3) get 5mL fibrin ferment mother liquor, join the whole 1L of residue and protect in liquid, dilution ratio is about 1:200, obtains 1L thrombin reagent, and mixing is thrombin time (TT) and detects reagent.
5 thrombin times (TT) reagent Performance Detection:
1) accuracy detects
On above-mentioned thrombin reagent, Sysmex CA-1500 coagulo meter is fresh (2h in) plasma sample normally to 30 routine hospital reports, and detection method is the same.Result is as following table 1:
Table 1 thrombin time detects reagent test result
| Sample | Result | Sample | Result |
| Blood plasma 1 | 11.4 | Blood plasma 16 | 13.3 |
| Blood plasma 2 | 13.7 | Blood plasma 17 | 15.3 |
| Blood plasma 3 | 17.9 | Blood plasma 18 | 16.1 |
| Blood plasma 4 | 13.3 | Blood plasma 19 | 15.4 |
| Blood plasma 5 | 14.5 | Blood plasma 20 | 15.8 |
| Blood plasma 6 | 17.5 | Blood plasma 21 | 16.8 |
| Blood plasma 7 | 14.1 | Blood plasma 22 | 15.5 |
| Blood plasma 8 | 13.4 | Blood plasma 23 | 16.1 |
| Blood plasma 9 | 11.4 | Blood plasma 24 | 18.9 |
| Blood plasma 10 | 13.4 | Blood plasma 25 | 16.1 |
| Blood plasma 11 | 19.2 | Blood plasma 26 | 15.8 |
| Blood plasma 12 | 13.7 | Blood plasma 27 | 14.6 |
| Blood plasma 13 | 13.9 | Blood plasma 28 | 15.9 |
| Blood plasma 14 | 15.8 | Blood plasma 29 | 13.5 |
| Blood plasma 15 | 13.9 | Blood plasma 30 | 19.7 |
Above-mentioned normal sample results all in the 20s of Industry code requirements reagent accuracy meet the requirements.
2) consistency detection between bottle
Often being propped up by 4mL by the thrombin time of preparation detection reagent is sub-packed in 7mL clear glass bottle, randomly draw 5 to go up Sysmex CA1500 coagulo meter respectively and detect Siemens blood clotting normal level quality-control product Ci-Trol 1 and fresh normal plasma, each 1 routine sample of abnormal plasma, result is as table 2:
Table 2 thrombin time detects uniformity test result between reagent bottle
| Sample (s) | Bottle 1 | Bottle 2 | Bottle 3 | Bottle 4 | Bottle 5 | CV |
| Ci-Trol 1 | 13.7 | 13.5 | 13.7 | 13.7 | 13.5 | 0.80％ |
| Normal plasma | 14.3 | 14.7 | 14.5 | 14.6 | 14.5 | 1.02％ |
| Abnormal plasma | 23.7 | 23.5 | 23.8 | 23.7 | 23.2 | 1.01％ |
The consistance that the above results shows 5 bottles of each samples of reagent test is all better, and between bottle, difference is within 2%.
Embodiment 2 thrombin time detects the heat stability test of reagent
1 preparation of reagents: the formulated in combination various fibrin ferment protection liquid of according to the form below 3, then with 1:200 dilution proportion fibrin ferment mother liquor, obtains each thrombin time and detect reagent.
The combination of table 3 each fibrin ferment protection liquid composition
237 DEG C of heat stability test: each thrombin time of above-mentioned preparation is detected reagent and contrast agent 1 parallel experiment, often propping up by 4mL is sub-packed in 7mL clear glass bottle, seal, put in 37 DEG C of baking ovens for 30 and carry out heat damage, respectively get one every day to detect the Siemens blood clotting normal level quality-control product Ci-Trol 1 of the new same lot number redissolved, detection method is the same.Result is as following table 4:
Table 4 thrombin time detects reagent 37 DEG C of heat stability test results (unit: second)
Upper table result display, 37 DEG C of heat damages after 30 days, thrombin time in the present invention detects reagent (reagent 1 ~ reagent 4) testing result to sample and all offsets within 1.1s (10%), illustrates that reagent can stablize more than 30 days under 37 DEG C of high temperature; And contrast agent 37 DEG C place 10 days afterwards skew namely more than 1.6s (being greater than 10%), show 37 DEG C and stablize at most 10 days.
The long-time stability that embodiment 3 thrombin time detects reagent detect
1: preparation of reagents: prepare fibrin ferment protection liquid by combination 150mmol/L Tris, 3% glycocoll, 0.7%PEG20000,8%BSA, 0.3% gelatin, 5% sweet mellow wine, 0.1% Sodium azide, pH7.4; then press 1:200 dilution proportion fibrin ferment mother liquor, obtain fibrin ferment and detect reagent (being designated as reagent 5).For display effect, parallel experiment is carried out with contrast agent 2, the component of contrast agent 2 is: 50mM pH7.5Tris-HCl, 0.5%PEG20000,0.25%BSA, 3% glycerine, 0.1% hydroxybutyric acid salt toluene, 0.1% Sodium azide, 40mM sodium chloride, 0.1% gelatin, thrombin of beef final concentration 5IU/mL.
2: long-time stability detect: reagent 5 and contrast agent 2 are often propped up by 4mL respectively and be sub-packed in 7mL clear glass bottle, seal, put the storage of 2 ~ 8 DEG C, refrigerator for 20, monthly within 15th, respectively get one to detect the new same lot number Siemens blood clotting normal level quality-control product Ci-Trol 1 redissolved, detection method is the same.Result is as following table 5:
Table 5 thrombin time detects reagent long-time stability testing result (unit: second)
Upper table result display, in 2 ~ 8 DEG C store 18 months, the liquid thrombin reagent in the present invention within 1s (10%), illustrates that reagent stores the term of validity and can reach more than 18 months to the testing result skew of sample; And contrast agent 2 at 14 months hour offsets namely more than 1.7s (being greater than 10%).Show this reagent extended storage stability significantly to promote.
At present, commercially available thrombin time detects the reagent that reagent mostly is freeze-dried type, and after preparation of reagents is good, freeze-drying is dry powder, to keep the activity of fibrin ferment, before detecting, needs the redissolution that adds water.
Amino acid, particularly glycocoll or alanine; Polyglycol, the particularly polyglycol of molecular weight 8000-20000; Sweet mellow wine; Bovine serum albumin(BSA) and gelatin, these component compatibilities, can keep the stability of fibrin ferment in liquid reagent well.And be 1% ~ 5% glycocoll or alanine containing quality percent by volume; Quality percent by volume is the PEG 8000-2000 of 0.4% ~ 1%; Quality percent by volume is the sweet mellow wine of 3% ~ 10%; Quality percent by volume is the bovine serum albumin(BSA) of 3% ~ 12%; Quality percent by volume is the gelatin of 0.1% ~ 0.5%, and pH is that the performance of the liquid-type reagent of 6-8 can meet commercial requirement completely.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. liquid thrombin time detects a reagent, and it is characterized in that, containing fibrin ferment, amino acid, polyglycol, sweet mellow wine, bovine serum albumin(BSA), gelatin, and solvent is water.
2. liquid thrombin time according to claim 1 detects reagent, and it is characterized in that, its pH value is 6.0 ~ 8.0.
3. liquid thrombin time according to claim 1 detects reagent, and it is characterized in that, described amino acid is selected from glycocoll or alanine.
4. liquid thrombin time according to claim 1 detects reagent, and it is characterized in that, the molecular weight ranges of described polyglycol is at 8000-20000.
5. liquid thrombin time according to claim 1 detects reagent, and it is characterized in that, the enzymatic activity of described fibrin ferment is 5 ~ 80IU/mL.
6. liquid thrombin time as claimed in any of claims 1 to 5 detects reagent, it is characterized in that, also comprises buffer salt.
7. liquid thrombin time according to claim 6 detects reagent, it is characterized in that, also comprises sodium azide.
8. liquid thrombin time according to claim 7 detects reagent, and it is characterized in that, described amino acid whose quality percent by volume is 1% ~ 5%;
The quality percent by volume of polyglycol is 0.4% ~ 1%;
The quality percent by volume of sweet mellow wine is 3% ~ 10%;
The quality percent by volume of bovine serum albumin(BSA) is 3% ~ 12%;
The quality percent by volume of gelatin is 0.1% ~ 0.5%.
9. liquid thrombin time detects a preparation method for reagent, it is characterized in that, comprising:
Amino acid, polyglycol, sweet mellow wine, bovine serum albumin(BSA), Gelatin are obtained in water fibrin ferment protection liquid, adjust ph 6.0 ~ 8.0;
Get fibrin ferment freeze-dried powder to be dissolved in described fibrin ferment protection liquid, obtain described liquid thrombin time and detect reagent, the enzymatic activity that described liquid thrombin time detects fibrin ferment in reagent is 5 ~ 80IU/mL.
10. preparation method as claimed in claim 9, is characterized in that, gets fibrin ferment freeze-dried powder and is dissolved in described fibrin ferment protection liquid, obtains the step that described liquid thrombin time detects reagent and comprises:
Get fibrin ferment freeze-dried powder be dissolved in described fibrin ferment protection liquid in obtain fibrin ferment mother liquor, in described fibrin ferment mother liquor, the enzymatic activity of fibrin ferment is 3000 ~ 10000IU/mL;
The described fibrin ferment protection liquid of described fibrin ferment mother liquor is diluted respectively, obtains some dilutability solution and titration, choose a dilutability according to titration results;
According to described dilutability, dilute described fibrin ferment mother liquor with described fibrin ferment protection liquid, obtain described liquid thrombin time and detect reagent.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105424946A (en) * | 2015-07-16 | 2016-03-23 | 青岛古高生物科技有限公司 | Rosmarinic acid-containing thromboplastin time measuring reagent |
| 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 |
| WO2018227359A1 (en) * | 2017-06-13 | 2018-12-20 | Chung Chin Sun | A novel method for blood serum protein activity preservation |
| CN111638375A (en) * | 2020-06-08 | 2020-09-08 | 深圳市国赛生物技术有限公司 | In-vitro diagnostic kit for measuring activated partial thromboplastin time |
| CN114058675A (en) * | 2020-08-04 | 2022-02-18 | 深圳市帝迈生物技术有限公司 | Stabilizer, thrombin time testing reagent, preparation method and kit thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105424946A (en) * | 2015-07-16 | 2016-03-23 | 青岛古高生物科技有限公司 | Rosmarinic acid-containing thromboplastin time measuring reagent |
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| WO2018227359A1 (en) * | 2017-06-13 | 2018-12-20 | Chung Chin Sun | A novel method for blood serum protein activity preservation |
| 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 |
| CN111638375A (en) * | 2020-06-08 | 2020-09-08 | 深圳市国赛生物技术有限公司 | In-vitro diagnostic kit for measuring activated partial thromboplastin time |
| CN111638375B (en) * | 2020-06-08 | 2022-12-13 | 深圳市国赛生物技术有限公司 | In-vitro diagnostic kit for measuring activated partial thromboplastin time |
| CN114058675A (en) * | 2020-08-04 | 2022-02-18 | 深圳市帝迈生物技术有限公司 | Stabilizer, thrombin time testing reagent, preparation method and kit thereof |
| CN114058675B (en) * | 2020-08-04 | 2022-11-15 | 深圳市帝迈生物技术有限公司 | Stabilizer, thrombin time testing reagent, preparation method thereof and kit |
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