CN109239373A - A kind of tissue factor carboxylate reagent and the preparation method and application thereof - Google Patents
A kind of tissue factor carboxylate reagent and the preparation method and application thereof Download PDFInfo
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- 108010000499 Thromboplastin Proteins 0.000 title claims abstract description 104
- 102000002262 Thromboplastin Human genes 0.000 title claims abstract description 104
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 69
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 150000007942 carboxylates Chemical class 0.000 title abstract 4
- 150000003904 phospholipids Chemical class 0.000 claims abstract description 50
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 33
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 33
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims abstract description 30
- 239000003381 stabilizer Substances 0.000 claims abstract description 19
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 claims abstract description 15
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 claims abstract description 15
- 235000012000 cholesterol Nutrition 0.000 claims abstract description 15
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims abstract description 15
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 claims abstract description 12
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 claims abstract description 12
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 claims abstract description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 11
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 claims abstract description 11
- 229940114124 ferulic acid Drugs 0.000 claims abstract description 11
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 claims abstract description 11
- 235000001785 ferulic acid Nutrition 0.000 claims abstract description 11
- 239000008103 glucose Substances 0.000 claims abstract description 11
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000000872 buffer Substances 0.000 claims abstract description 8
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 6
- 230000032050 esterification Effects 0.000 claims description 31
- 238000005886 esterification reaction Methods 0.000 claims description 31
- 239000000126 substance Substances 0.000 claims description 30
- 239000011259 mixed solution Substances 0.000 claims description 22
- 238000002156 mixing Methods 0.000 claims description 21
- 239000004094 surface-active agent Substances 0.000 claims description 16
- 239000007853 buffer solution Substances 0.000 claims description 15
- 239000003755 preservative agent Substances 0.000 claims description 13
- 230000002335 preservative effect Effects 0.000 claims description 12
- 238000000502 dialysis Methods 0.000 claims description 11
- 229940080421 coco glucoside Drugs 0.000 claims description 8
- 239000007983 Tris buffer Substances 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical group [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 108010094028 Prothrombin Proteins 0.000 claims description 4
- 102100027378 Prothrombin Human genes 0.000 claims description 4
- MRUAUOIMASANKQ-UHFFFAOYSA-N cocamidopropyl betaine Chemical compound CCCCCCCCCCCC(=O)NCCC[N+](C)(C)CC([O-])=O MRUAUOIMASANKQ-UHFFFAOYSA-N 0.000 claims description 4
- 229940073507 cocamidopropyl betaine Drugs 0.000 claims description 4
- 229940079988 potassium cocoyl glycinate Drugs 0.000 claims description 4
- 229940039716 prothrombin Drugs 0.000 claims description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 3
- 239000007995 HEPES buffer Substances 0.000 claims description 3
- -1 n-octyl Chemical group 0.000 claims description 3
- 238000004321 preservation Methods 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 2
- 238000005215 recombination Methods 0.000 abstract 2
- 230000006798 recombination Effects 0.000 abstract 2
- 239000000203 mixture Substances 0.000 description 14
- 206010053567 Coagulopathies Diseases 0.000 description 7
- 230000035602 clotting Effects 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000009472 formulation Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 238000003149 assay kit Methods 0.000 description 2
- 230000015271 coagulation Effects 0.000 description 2
- 238000005345 coagulation Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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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
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Abstract
The present invention relates to a kind of tissue factor carboxylate reagents and the preparation method and application thereof, belong to technical field of in vitro diagnostic reagents.It solves and how the technical issues of a kind of at low cost, stability is good, tissue factor carboxylate reagent with long preservation period and the preparation method and application thereof is provided.Reagent of the invention, including recombination rabbit tissue factor, synthetic phospholipid, stabilizer and buffer；Wherein, concentration of the recombination rabbit tissue factor in reagent is 1 μ g/ml；Synthetic phospholipid is made of phosphatidylserine, phosphatidyl choline, phosphatidyl glycerol and cholesterol, and concentration of the synthetic phospholipid in reagent is 250 μ g/ml；Stabilizer is made of glycerol, glucose and ferulic acid.Type, proportion of the tissue factor carboxylate reagent by adjusting synthetic phospholipid, stabilizer and buffer, have that at low cost, stability is high, advantage with long preservation period.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnostic reagent raw materials, and particularly relates to a tissue factor esterified substance reagent and a preparation method and application thereof.
Background
Tissue factor is a protein. At present, genetic engineering means are adopted at home and abroad to express and produce tissue factors in escherichia coli, fungi or animal cells. The chinese patent publication No. 101294163B successfully expresses human recombinant tissue factor in yeast by using genetic engineering means. The method is more efficient and has higher product purity than conventional method for extracting tissue factor from animal tissue. Tissue factor can be mixed with phospholipid in a certain proportion to form tissue factor esterified substance. In the esterified product, tissue factor is embedded into the liposome surface formed by phospholipid bilayer. Tissue factor esters are effective in activating the extrinsic coagulation pathway and are often used in the preparation of Prothrombin Time (PT) assay kits. Conventionally, tissue factor esterified substance is often extracted from animal tissue, such as PT reagent of Shanghai Sun company which extracts tissue factor esterified substance from rabbit brain tissue, and Siemens company which extracts tissue factor esterified substance from human placenta. The naturally extracted tissue factor already contains phospholipids, so that no additional phospholipids are required. The naturally extracted tissue factor esterified substance is susceptible to various factors, such as the age, sex, diet, individual difference, etc. of animals or humans, which greatly affect the content of the tissue factor and the content and composition of phospholipid. The detection effect of different tissue factor contents on the PT reagent is obviously different due to different phospholipid compositions and contents. This is also one of the main reasons for the large lot-to-lot variation of the PT assay kit. The tissue factor esterified substance prepared by mixing the tissue factor prepared by adopting a genetic engineering means and a certain proportion of phospholipid has definite components, and the process is controllable, so that the batch difference of the PT detection kit can be effectively reduced. At present, the tissue factor ester manufacturers in China have Taiyuan Boott, Qingdao Kangda and the like. However, the price of the commercial product is higher, which limits the popularization and application.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a tissue factor esterification reagent and a preparation method and application thereof, and the tissue factor esterification reagent has the advantages of low cost, good stability and long-term storage.
The technical scheme adopted by the invention for solving the technical problems is as follows.
The invention provides a tissue factor esterification reagent, which comprises recombinant rabbit tissue factor, synthetic phospholipid and buffer solution containing a stabilizing agent;
the concentration of the recombinant rabbit tissue factor in the reagent is 1 mug/ml;
the synthetic phospholipid consists of phosphatidylserine, phosphatidylcholine, phosphatidylglycerol and cholesterol, the mass ratio of the phosphatidylserine to the phosphatidylcholine to the phosphatidylglycerol to the cholesterol is 3:6:0:1, 3:3:3:1, 3:0:3:4 or 0:3:3:4, and the concentration of the synthetic phospholipid in a reagent is 250 mu g/ml;
the stabilizing agent consists of glycerol, glucose and ferulic acid, wherein the concentration of the glycerol, the glucose and the ferulic acid in the buffer solution is 10%, 5%, 0.02%, or 12%, 5%, 0.02%, or 7%, 3%, 0.02%, or 10%, 5%, 0.01%, or 12%, 5%, 0.01% in sequence.
Preferably, the agent further comprises 0.02% -0.1% of a preservative.
More preferably, the concentration of the preservative is 0.05%.
Preferably, the preservative is sodium azide and/or Proclin 300.
Preferably, the buffer is PBS buffer, Tris buffer or HEPES buffer.
More preferably, the buffer is at a concentration of 20 mM.
The invention also provides a preparation method of the tissue factor esterified reagent, which comprises the following steps:
mixing phosphatidylserine, phosphatidylcholine, phosphatidylglycerol and cholesterol according to a ratio to obtain synthetic phospholipid;
step two, mixing the synthetic phospholipid and the surfactant, and completely dissolving the synthetic phospholipid after uniformly mixing to obtain a first mixed solution, wherein the concentration of the synthetic phospholipid in the first mixed solution is 35 mg/ml;
adding the recombinant rabbit tissue factor into the first mixed solution, uniformly mixing, stirring and incubating at room temperature for 4 hours to obtain a second mixed solution with the concentration of the recombinant rabbit tissue factor of 80-120 mu g/ml, and removing the surfactant in the second mixed solution by a dialysis mode to obtain a recombinant rabbit tissue factor esterified substance;
and step four, diluting the recombinant rabbit tissue factor esterified substance obtained in the step three by using a buffer solution containing a stabilizing agent to obtain the tissue factor esterified substance reagent.
Preferably, in step two, the surfactant is potassium cocoyl glycinate, coco glucoside, cocamidopropyl betaine or n-octyl glucopyranoside.
Preferably, in step three, the dialysis method is as follows: the second mixed solution was transferred to a dialysis bag and dialyzed once every 4 hours for a total of five times to remove the surfactant.
The invention also provides application of the tissue factor esterified reagent in preparation of a prothrombin time detection kit.
Compared with the prior art, the invention has the beneficial effects that:
1. the tissue factor esterified reagent has the advantages of low cost, high stability and long-term preservation by adjusting the types and the proportion of the synthetic phospholipid, the stabilizing agent and the buffer solution, and specifically comprises the following components in percentage by weight:
the tissue factor esterified substance reagent has clear components, and overcomes the technical problems of unclear type and content of naturally extracted tissue factor esterified substances and large batch difference in the prior art;
the tissue factor esterification reagent adopts phosphatidylserine, phosphatidylcholine, phosphatidylglycerol and cholesterol as synthetic phospholipid and adopts glycerol, glucose and ferulic acid as stabilizing agents, so that the stability of the tissue factor esterification can be effectively improved.
2. The tissue factor esterified substance reagent can be directly used as a PT detection reagent, and provides a basis for popularization and application of the tissue factor esterified substance.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention, but it is to be understood that the description is intended to illustrate further features and advantages of the invention, and not to limit the scope of the claims.
The invention provides a tissue factor esterified reagent, which comprises recombinant rabbit tissue factor, synthetic phospholipid, a stabilizer and a buffer solution; wherein,
the concentration of the recombinant rabbit tissue factor in the reagent is 1 mug/ml;
the synthetic phospholipid consists of phosphatidylserine, phosphatidylcholine, phosphatidylglycerol and cholesterol, the mass ratio of the phosphatidylserine to the phosphatidylcholine to the phosphatidylglycerol to the cholesterol is 3:6:0:1, 3:3:3:1, 3:0:3:4 or 0:3:3:4, preferably the synthetic phospholipid consists of the phosphatidylserine, the phosphatidylcholine and the cholesterol in a mass ratio of 3:6:1, and the concentration of the synthetic phospholipid in the reagent is 250 mug/ml;
the stabilizer consists of glycerol, glucose and ferulic acid, wherein the concentration of the glycerol, the glucose and the ferulic acid in the buffer solution is 10%, 5%, 0.02%, or 12%, 5%, 0.02%, or 7%, 3%, 0.02%, or 10%, 5%, 0.01%, or 12%, 5%, 0.01% in sequence.
In the above technical scheme, in order to further enhance the stability of the reagent, the tissue factor esterification reagent may further include a preservative, and the concentration of the preservative is 0.02% to 0.1%, preferably 0.05%; the preservative is preferably sodium azide and/or Proclin 300.
In the above technical scheme, the buffer solution is a buffer solution commonly used in the art, such as PBS buffer solution, Tris buffer solution or HEPES buffer solution, and the concentration is preferably 20 mM.
The invention also provides a preparation method of the tissue factor esterified reagent, which comprises the following steps:
mixing phosphatidylserine, phosphatidylcholine, phosphatidylglycerol and cholesterol according to the mass ratio of 3:6:3:1, 3:3:3:1, 3:0:3:4 or 0:3:3:4 to obtain synthetic phospholipid;
step two, mixing the synthetic phospholipid and the surfactant, and completely dissolving the synthetic phospholipid after uniformly mixing to obtain a first mixed solution, wherein the concentration of the synthetic phospholipid in the first mixed solution is 35 mg/ml;
among them, the surfactant is preferably potassium cocoyl glycinate, coco glucoside, cocamidopropyl betaine or n-octyl glucopyranoside, and particularly preferably coco glucoside;
adding the recombinant rabbit tissue factor into the first mixed solution, uniformly mixing, stirring and incubating at room temperature for 4 hours to obtain a second mixed solution with the concentration of the recombinant rabbit tissue factor of 80-120 mu g/ml, and removing the surfactant in the second mixed solution by a dialysis mode to obtain a recombinant rabbit tissue factor esterified substance;
wherein, the dialysis mode is as follows: transferring the second mixed solution into a dialysis bag, dialyzing once every 4 hours for five times in total, and removing the surfactant;
step four, diluting the synthesized recombinant rabbit tissue factor esterified substance by using a buffer solution containing a stabilizing agent until the synthesized recombinant rabbit tissue factor esterified substance contains 1 microgram/ml of recombinant rabbit tissue factor and 250 microgram/ml of synthetic phospholipid, namely a tissue factor esterified substance reagent;
wherein the stabilizer consists of glycerol, glucose and ferulic acid, and the concentration of the glycerol, the glucose and the ferulic acid in the buffer solution is 10%, 5%, 0.02%, or 12%, 5%, 0.02%, or 7%, 3%, 0.02%, or 10%, 5%, 0.01%, or 12%, 5%, 0.01%;
when the tissue factor esterified reagent contains the preservative, the preservative is added together with the buffer solution containing the stabilizing agent according to the mixture ratio in the fourth step.
The invention also provides application of the tissue factor esterified reagent in preparation of a prothrombin time detection kit.
The present invention is further illustrated by the following examples.
Example 1
Preparing a tissue factor esterified reagent, which comprises the following steps;
step one, weighing phosphatidylserine, phosphatidylcholine, phosphatidylglycerol and cholesterol in each formula respectively according to the proportion in table 1, and uniformly mixing to obtain synthetic phospholipid;
step two, mixing the synthetic phospholipid with a surfactant, and completely dissolving the synthetic phospholipid after uniformly mixing to obtain a mixed solution, wherein the final concentration of the synthetic phospholipid is 35 mg/ml;
step three, adding recombinant rabbit tissue factors into the mixed solution of each formula respectively according to the proportion of table 1, wherein the final concentration of the recombinant rabbit tissue factors is 120 mug/ml, uniformly mixing, stirring and incubating at room temperature for 4 hours, transferring the obtained solution into a dialysis bag, dialyzing once every 4 hours for five times, and removing the surfactant to obtain a recombinant rabbit tissue factor esterified substance;
step four, diluting the recombinant rabbit tissue factor esterified substance obtained in the step three by using a Tris buffer solution containing 10% of glycerol, 5% of glucose and 0.02% of ferulic acid to obtain a tissue factor esterified substance reagent containing 1 mu g/ml of recombinant rabbit tissue factor and 250 mu g/ml of synthetic phospholipid.
TABLE 1 compositions and ratios of formulations used in tissue factor esterification reagents of example 1
In Table 1, the percentage indicates the mass percentage of the substance in the synthetic phospholipid.
The results of measuring the clotting time of normal plasma using the tissue factor esterification reagent of example 1 are shown in Table 2.
TABLE 2 measurement of clotting time of Normal plasma with tissue factor esterification reagent of example 1
As seen from Table 2, the results of the measurement of the tissue factor esterification reagents prepared in the formulae 1 to 4 in Table 1 were all in the normal range, wherein the formula 1 is close to the middle of the reference interval, indicating that the synthetic phospholipid is preferably composed of phosphatidylserine, phosphatidylcholine and cholesterol in a mass ratio of 3:6: 1.
Example 2
Synthetic tissue factor esterification reagents (see table 3 for composition and ratio of specific formulation) were prepared by replacing the surfactants in formula 1 of example 1 with potassium cocoyl glycinate, cocoyl glucoside and cocamidopropyl betaine, respectively, and the rest was the same as in example 1.
TABLE 3 composition and ratio of the formulation used in tissue factor esterification reagent of example 2
In Table 2, the percentage indicates the mass percentage of the substance in the synthetic phospholipid.
The results of measuring the clotting time of normal plasma using the tissue factor esterification reagent of example 2 are shown in Table 4.
TABLE 4 measurement of clotting time of Normal plasma with tissue factor esterification reagent of example 2
As seen from Table 4, the results of the tissue factor esterification reagents prepared from the recipes 1, 5, 6 and 7 in Table 3 were in the normal range, where the recipe 1 was close to the middle of the reference interval. It is preferred that coco glucoside be used as a surfactant in the present invention.
Example 3
The preparation of tissue factor ester reagent comprises the following steps:
step one, mixing phosphatidylserine, phosphatidylcholine and cholesterol according to a mass ratio of 3:6:1 to obtain synthetic phospholipid;
step two, mixing the synthetic phospholipid and the coco-glucoside solution, and after uniformly mixing the synthetic phospholipid and the coco-glucoside solution to completely dissolve the synthetic phospholipid, obtaining a mixed solution, wherein the final concentration of the synthetic phospholipid is 35 mg/ml;
adding the recombinant rabbit tissue factor into the mixed solution, wherein the final concentration of the recombinant rabbit tissue factor is 120 mu g/ml, uniformly mixing, stirring and incubating at room temperature for 4h, and removing coco glucoside by adopting a dialysis mode to obtain a recombinant rabbit tissue factor esterified substance;
step four, according to the mixture ratio of the table 5, respectively diluting the recombinant rabbit tissue factor esterified substance obtained in the step three by using Tris buffer solutions containing different stabilizing agents until the recombinant rabbit tissue factor containing 1 microgram/ml and the synthetic phospholipid containing 250 microgram/ml are obtained, namely the tissue factor esterified substance reagent.
TABLE 5 composition of stabilizers in different stabilizer-containing Tris buffers in example 3
In Table 5, the percentage indicates the mass percentage of the substance in the Tris buffer.
The results of measuring the clotting time of normal plasma using the tissue factor esterification reagent of example 3 are shown in Table 6.
TABLE 6 measurement of clotting time of Normal plasma with tissue factor esterification reagent of example 3
As is apparent from Table 6, the measurement results of the tissue factor esterification reagents prepared from the tissue factor esterifiers prepared in the recipes 1, 9, 11, 15 and 16 in Table 5 are in the normal range.
Example 4
Different kinds of preservatives were added to the tissue factor esterification reagent of example 1 in different amounts according to the formulation of table 7.
TABLE 7 antiseptic composition and formulation ratio of tissue factor esterification reagent of example 4
In Table 7, the percentage indicates the mass percentage of the preservative in the tissue factor esterification reagent.
The results of measuring the clotting time of the quality control products after the reagents of example 4 were left at 37 ℃ for 7 days are shown in Table 8.
TABLE 8 measurement of coagulation time of quality control product by tissue factor esterification reagent in example 4
As can be seen from Table 8, the measurement results of the tissue factor esterification reagents prepared in the formulas 17 to 22 are all in the reference range.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The tissue factor esterified reagent is characterized by comprising recombinant rabbit tissue factor, synthetic phospholipid, a stabilizer and a buffer solution;
the concentration of the recombinant rabbit tissue factor in the reagent is 1 mug/ml;
the synthetic phospholipid consists of phosphatidylserine, phosphatidylcholine, phosphatidylglycerol and cholesterol, the mass ratio of the phosphatidylserine to the phosphatidylcholine to the phosphatidylglycerol to the cholesterol is 3:6:0:1, 3:3:3:1, 3:0:3:4 or 0:3:3:4, and the concentration of the synthetic phospholipid in a reagent is 250 mug/ml;
the stabilizing agent consists of glycerol, glucose and ferulic acid, wherein the concentration of the glycerol, the glucose and the ferulic acid in the buffer solution is 10%, 5%, 0.02%, or 12%, 5%, 0.02%, or 7%, 3%, 0.02%, or 10%, 5%, 0.01%, or 12%, 5%, 0.01% in sequence.
2. The tissue factor esterification reagent according to claim 1, further comprising 0.02 to 0.1% of a preservative.
3. The tissue factor esterification reagent according to claim 2, wherein the concentration of the preservative is 0.05%.
4. The tissue factor esterification reagent according to claim 2, wherein the preservative is sodium azide and/or Proclin 300.
5. The tissue factor esterification reagent according to claim 1, wherein the buffer is a PBS buffer, a Tris buffer, or a HEPES buffer.
6. The tissue factor esterification reagent according to claim 1, wherein the buffer has a concentration of 20 mM.
7. The method for preparing the tissue factor esterification reagent according to any one of claims 1 to 6, comprising the steps of:
mixing phosphatidylserine, phosphatidylcholine, phosphatidylglycerol and cholesterol according to a ratio to obtain synthetic phospholipid;
step two, mixing the synthetic phospholipid and the surfactant, and completely dissolving the synthetic phospholipid after uniformly mixing to obtain a first mixed solution, wherein the concentration of the synthetic phospholipid in the first mixed solution is 35 mg/ml;
adding the recombinant rabbit tissue factor into the first mixed solution, uniformly mixing, stirring and incubating at room temperature for 4 hours to obtain a second mixed solution with the concentration of the recombinant rabbit tissue factor of 80-120 mu g/ml, and removing the surfactant in the second mixed solution by a dialysis mode to obtain a recombinant rabbit tissue factor esterified substance;
and step four, diluting the recombinant rabbit tissue factor esterified substance obtained in the step three by using a buffer solution containing a stabilizing agent to obtain the tissue factor esterified substance reagent.
8. The method of claim 7, wherein in step two, the surfactant is potassium cocoyl glycinate, coco glucoside, cocamidopropyl betaine, or n-octyl glucopyranoside.
9. The method for preparing a tissue factor esterification reagent according to claim 7, wherein the dialysis method in step three is: the second mixed solution was transferred to a dialysis bag and dialyzed once every 4 hours for a total of five times to remove the surfactant.
10. Use of the tissue factor esterification reagent according to any one of claims 1 to 6 for the preparation of a prothrombin time detection kit.
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| CN201810927731.9A CN109239373A (en) | 2018-08-15 | 2018-08-15 | A kind of tissue factor carboxylate reagent and the preparation method and application thereof |
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| US5314695A (en) * | 1990-11-13 | 1994-05-24 | Corvas International, Inc. | Tissue factor based prothrombin time reagent |
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| CN104914256A (en) * | 2014-03-13 | 2015-09-16 | 基立福有限公司 | Composition for use as an abnormal coagulation control plasma in in vitro assays |
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