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CN102532316A - Anti-vWF (von Willebrand factor) monoclonal antibody and application thereof - Google Patents

Anti-vWF (von Willebrand factor) monoclonal antibody and application thereof Download PDF

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CN102532316A
CN102532316A CN2010106036672A CN201010603667A CN102532316A CN 102532316 A CN102532316 A CN 102532316A CN 2010106036672 A CN2010106036672 A CN 2010106036672A CN 201010603667 A CN201010603667 A CN 201010603667A CN 102532316 A CN102532316 A CN 102532316A
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vwf
antibody
seq
coagulation factor
blood coagulation
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CN102532316B (en
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谢良志
盖文琳
张建东
孙春昀
罗春霞
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SHENZHOU CELL ENGINEERING Co Ltd
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Abstract

The invention provides an anti-vWF (von Willebrand factor) monoclonal antibody. The antibody can perform specific binding with human vWF and can also bind with a compound of vWF and a coagulation factor VIII. The monoclonal antibody is immobilized on a chromatography medium so as to prepare an affinity medium, and the affinity medium can be used for purifying vWF and the coagulation factor VIII.

Description

A kind of anti-vWF monoclonal antibody and application thereof
Technical field
The present invention relates to biological technical field, specifically, the present invention relates to a kind of anti-vWF (von Willebrand factor) monoclonal antibody of high-affinity, this antibody capable and people vWF specific combination also can combine with the complex body of vWF and blood coagulation factor VIII.Also relate to a kind of method and a kind of method of utilizing this antibody purification blood coagulation factor VIII of utilizing this antibody purification vWF simultaneously.
Background technology
Coagulation process is the process of a complicacy, and it comprises a series of biochemical reaction, relates to more than ten kind of thrombin.The disappearance of any thrombin or function variation all can cause the blood coagulation relative disease.Hemophilia is exactly a kind of shortage blood coagulation factor VIII or vWF (von Willebrand factor) and the hemorrhagic diseases that causes.
VWF is present in the blood plasma, and it is the carrier of blood coagulation factor VIII, can stablize blood coagulation factor VIII, and prevents that blood coagulation factor VIII is by protease hydrolysis.VWF is a kind of adhesion gp simultaneously, in hematoblastic sticking, plays central role.Through the adhesion of thrombocyte, start coagulation process in damaged vessel walls.The polymer that vWF is made up of the 230kD subunit, its molecular weight is from 5 * 10 5To 10 7Da.VWF contains the sugar of 5-6%, and these sugar combine hematoblastic function extremely important for vWF.VWF is synthetic in endotheliocyte (endothelial cell) and megalokaryocyte (megakaryochtes).And through disulfide linkage formation dimer.Be that the basis is connected to form high polymer again with the aggressiveness.High-molecular weight vWF polymer is extremely important in coagulation process.Various vWF activity can cause von Willebrand syndromes unusually.Too much vWF can increase the risk of thrombus, and the vWF level is crossed and low can be caused hemorrhage tendency, owing to suppressed hematoblastic gathering, the bleeding time after injured prolongs.The shortage of vWF also can show the symptom of hemophilia A; This is because vWF stable extremely important for blood coagulation factor VIII; The shortage of vWF causes the transformation period of blood coagulation factor VIII to shorten, thereby the activity of blood coagulation factor VIII in the blood is reduced and shows the symptom of hemophilia A.The ordinary method of treatment von Willebrand syndromes is with the vWF that extracts in the blood plasma, also can use the vWF or the vWF-FVIII mixture of purifying.EP-A-0503991 and WO-A-8912065 provide the technology with ion-exchange purification vWF.US 6605222 and US 6579723 provide the technology for preparing the vWF-FVIII mixture.
Blood coagulation factor VIII is the important factor in the coagulation cascade reaction.It is the gp of a molecular weight 280kD, comprises two peptide chains of 200kD and 80kD.Blood coagulation factor VIII can speedup factor X after by activated by thrombin activation, finally form clot.Hemophilia A is exactly the chain disease of a kind of sex chromosome of the disappearance of blood coagulation factor VIII, account for ten thousand of male sex's population/.Haemophiliachemophiliac symptom is that injured back blood can not form blood clot or blood clot formation is very slow, causes haemophilia or bleeding time long.Haemophiliachemophiliac main treatment means is exactly supplement of coagulation factors VIII.In early days, adopt the way of input whole blood or blood plasma to come hemostasis for the hemophilia patient.But this method blood transfusion volume is big, and the blood coagulation factor VIII amount of input is also few, can only treat slight hemophilia patient.Can not effectively treat for serious hemophilia patient.A kind of cryoprecipitate legal systems of U.S. Pat 3803115 inventions in 1974 are equipped with the enriched material of blood coagulation factor VIII, and 29698 pairs of this method of US Re are further improved subsequently, have obtained active higher enriched material.Utilize the enriched material treatment hemophilia of blood coagulation factor VIII, can import more blood coagulation factor VIII, improved haemophiliachemophiliac result of treatment greatly.Though the specific activity of blood coagulation factor VIII increases substantially with comparing than blood plasma in the cryoprecipitate enriched material, but still contains a large amount of foreign proteins, spinoffs such as the immunoreation that causes behind the infusion are still very big, and infusion amount is also very big.Solvability in the cryoprecipitate enriched material after the stability of blood coagulation factor VIII and the freeze-drying of cryoprecipitate enriched material is also not ideal enough.So people are further purified blood coagulation factor VIII through various technique means on cryoprecipitate enriched material basis.US Re.32011 provides a kind of method of purifying blood coagulation Factor IX, and its process comprises the complex body of elder generation with affine absorption blood coagulation factor VIII of anti-vWF monoclonal antibody and vWF, and the wash-out blood coagulation factor VIII is used ammonia hexyl agarose purifying then again.The blood coagulation factor VIII specific activity of its preparation is compared with blood plasma and is improved 160000 times, and activity reaches 2300u/mg.Though the purity of the blood coagulation factor VIII that extracts from blood plasma at present is very high, exist the risk of infection transmissible disease.Along with the raising of gene engineering, the gene and the success that successfully clone blood coagulation factor VIII are now expressed in cell.Recombinant blood coagulation factor VIII compares tool with the blood coagulation factor VIII that blood plasma extracts and has great advantage: at first being the risk that has reduced blood-borne diseases, secondly is the restriction that does not receive the blood plasma source, can satisfy market demand by mass production.Once more, recombinant blood coagulation factor VIII product gas purity is high, and it is littler to compare the blood coagulation factor VIII spinoff of blood source.The blood coagulation factor VIII of reorganization has the trend that replaces blood plasma extraction blood coagulation factor VIII abroad.The blood coagulation factor VIII of at present commercially available reorganization has two kinds of the blood coagulation factor VIIIs that total length blood coagulation factor VIII and B district delete.Deletion back, B district blood coagulation factor VIII stability increases substantially, and helps the control in the production technique.But both not obviously differences of result of use.No matter be the total length blood coagulation factor VIII or the blood coagulation factor VIII of B district deletion; Its purge process all comprises the chromatography purification of a series of complicacies, but the production process of all commercial recombinant blood coagulation factor VIII has all comprised the immunoaffinity chromatography of anticoagulin VIII monoclonal antibody.The advantage of immunoaffinity chromatography is that cycles of concentration is high, good product purity.U.S. Pat 5597711 develops the affinity chromatography technology that replaces antibody with peptide again with US 5994310 recently.
Present anti-vWF antibody is widely used for the detection of vWF.U.S. Pat 5916805 and US6280731 provide one group of anti-vWF monoclonal antibody, can stop hematoblastic gathering, have the purposes of potential treatment thrombus.The avidity Kd of the anti-vWF antibody that U.S. Pat 6228360 provides is between 100~0.1nM.The invention provides a kind of anti-vWF monoclonal antibody, can combine with vWF also to combine, can be used for purifying vWF and also can be used for the purifying blood coagulation Factor IX with the complex body of vWF and blood coagulation factor VIII.The avidity Kd of anti-vWF antibody provided by the invention and vWF is 0.06nM.Avidity is higher than known same antibody-like.When being used for affinity purification, the antibody capable of high-affinity obtains high product purity.
Summary of the invention
The present invention provides a kind of anti-vWF mouse monoclonal antibody of high-affinity, it can with people vWF specific combination, also can combine with the complex body of vWF and blood coagulation factor VIII.This antibody is characterised in that: its light-chain amino acid sequence is SEQ ID NO:1, and heavy chain amino acid sequence is SEQ ID NO:2; Its light chain variable region amino acid sequence is SEQ ID NO:3, and its weight chain variable region amino acid sequence is SEQ ID NO:4; Complementary determining region CDR1 on the light chain, CDR2, the aminoacid sequence of CDR3 are respectively SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7; Complementary determining region CDR1 on the heavy chain, CDR2, the aminoacid sequence of CDR3 are respectively SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10.The avidity Kd of this antibody and vWF is 0.06nM.This antibody preparation process is following: adopting the Balb/c mouse as immune animal, is immunogen with recombinant human vWF albumen, and immunizing dose is 50 μ g/; First immunisation is processed emulsifying agent with the complete Freund's adjuvant of immunogen and equivalent, and subcutaneous abdomen multi-point injection, At intervals of two to three weeks get the same dose immunogen and the equivalent incomplete Freund's adjuvant is processed emulsifying agent; Serum titer is measured in twice, three immunity back of booster immunization, gets the high mouse peritoneal booster immunization of serum titer once; Get immune mouse spleen cell after 4 days; Merge in 4: 1 ratios and SP2/0 myeloma cell, adopt recombinant human vWF albumen, with ELISA method mensuration cell conditioned medium liquid as envelope antigen; Choose positive hole and carry out cloning, obtain the hybridoma cell strain of stably excreting vWF monoclonal antibody specific through limiting dilution.Select the cell strain of the required antibody of secretion, collect cultured cells, (Invitrogen) adds TRIzol to specifications, extracts total RNA, electrophoresis detection RNA quality, and UV measures concentration.Be reversed to cDNA by Roche reverse transcription test kit operation instructions ,-20 ℃ frozen subsequent use.Adopt the known technical scheme of professional domain personnel, reference (Ying W et al., BMCBioinformatics.2006; 7 (Suppl 4): S9)) design primer is the template light chain and the heavy chain fragment of PCR acquisition antibody respectively with cDNA.Light chain and heavy chain fragment TA to the pcDNA3T carrier that can be used for eukaryotic cell expression, are made up pcDNA3-anti-vWF-L and pcDNA3-anti-vWF-H expression vector.Transformed into escherichia coli, the picking positive colony, order-checking is identified.Analyze sequencing result, obtain correct light chain and heavy chain amino acid sequence.Murine hybridoma and antibody cloning technology are mature technologies, and process step is that this area scientific research personnel knows.
The present invention also provides a kind of method of utilizing this antibody purification vWF.At first will resist the monoclonal antibody of vWF to be fixed on and process affinity chromatography medium on the chromatography media.Let the feed liquid that contains vWF contact then with affinity chromatography medium, vWF promptly with affinity chromatography medium on the monoclonal antibody of anti-vWF combine.Other impurity of washing chromatography media flush away change solution condition then vWF and monoclonal antibody are dissociated, and promptly obtain the vWF of purifying.The used chromatography substrate of sessile antibody can be any activatory or non-activated chromatography substrate, includes but not limited to following material: Sepharose CL 4B, Separose 4 Fast Flow; Sepharose 6 Fast Flow, the Sepharose 4 Fast Flow of cyanogen bromide-activated, NHS activatory Sepharose; ECH Sepharose, EAHSepharose, Affi-Gel 10; Affi-Gel 15, Toyopearl-AF-Tresyl, Toyopearl-AF-Formyl; Toyopearl-AF-Carboxy, ProSep 5 CHO, ProSep 9 CHO etc.
Use the activatory chromatography substrate only to need to carry out antibody coupling according to supplier's specification sheets.Use non-activated chromatography substrate need carry out activation earlier, and then with antibody and the coupling of activatory chromatography substrate.Carrying out activation with cyanogen bromide is example, and the activatory key step is: Sepharose 4B is placed in the B and drains, and adds a spot of 0.1Mol/L pH 9.0NaHCO3 liquid washing; Under ice bath stirs, add cyanogen bromide, carefully drip 2Mol/LNaOH, pH is remained on about 11, reaction 10min.Adjust pH rapidly at 1min~2min and 8.0~11.0 keep 10min.The activatory agarose is poured into rapidly in the B, taken out with frozen water and wash neutrality, take out with cold 0.1Mol/L pH 9.0NaHCO3 rapidly again and wash.Promptly obtain activatory Sepharose.Matrix after the activation is identical with the use of activated substrate in advance, can be with reference to supplier's specification sheets.
The process of anti-vWF affinity chromatography medium absorption vWF must be carried out in the neutral environment of pH 6~8, and general cell culture fluid or serum need not adjusted pH or exchange buffering liquid, can directly adsorb.Will use the unconjugated impurity of neutral buffered liquid flush away of pH 6~8 after the absorption, buffer reagent can be selected any reagent that surge capability is arranged for use in neutral range, for example: Tris, HEPES, MES, phosphoric acid salt, imidazoles, Histidine etc.Wash-out vWF generally selects the acidic solution wash-out of pH2~3 for use, like the glycine buffer of pH3.0, also can select denaturing agent for use, for example: urea, Guanidinium hydrochloride, wash-outs such as Rhocya.With will with the Tris damping fluid damping fluid being adjusted to neutrality as early as possible behind the acidic solution wash-out, with utilizing ultrafiltration that vWF is replaced in the non-denaturing soln behind the denaturing agent wash-out as early as possible.It is that those skilled in the art grasp that affinity chromatography is separated, and can grasp flexibly according to specific circumstances.
The present invention also provides a kind of method of utilizing this antibody purification blood coagulation factor VIII.Blood coagulation factor VIII is to exist with the vWF bonding state in blood.In recombinant blood coagulation factor VIII produces, also can be with vWF and blood coagulation factor VIII coexpression, to improve the stability of blood coagulation factor VIII.The invention provides a kind of purification process easily for this with vWF bonded blood coagulation factor VIII.At first will resist the monoclonal antibody of vWF to be fixed on and process affinity chromatography medium on the chromatography media.Make feed liquid then and contact with affinity chromatography medium, the complex body of blood coagulation factor VIII and vWF promptly with affinity chromatography medium on the monoclonal antibody of anti-vWF combine.Other impurity of washing chromatography media flush away change solution condition then blood coagulation factor VIII and vWF are dissociated, and have just obtained purer blood coagulation factor VIII.Change solution condition wash-out vWF at last again and make affinity chromatography medium regeneration.The process of anti-vWF affinity chromatography medium absorption vWF/ blood coagulation factor VIII complex body must be carried out in the neutral environment of pH6~8, and general cell culture fluid or serum need not adjusted pH or exchange buffering liquid, can directly adsorb.Will use the unconjugated impurity of neutral buffered liquid flush away of pH 6~8 after the absorption, buffer reagent can be selected any reagent that surge capability is arranged for use in neutral range, for example: Tris, HEPES, MES, phosphoric acid salt, imidazoles, Histidine etc.Next step is to come the wash-out blood coagulation factor VIII through the vWF/ blood coagulation factor VIII complex body that dissociates.Dissociated solution is to contain 200~300mM CaCl 2The neutral buffered liquid of pH 6~8.VWF after dissociating also is combined on the affinity media.Last wash-out vWF generally selects the acidic solution wash-out of pH2~3 for use, like the glycine buffer of pH3.0, also can select denaturing agent for use, for example: urea, Guanidinium hydrochloride, wash-outs such as Rhocya.
Description of drawings
Fig. 1. anti-vWF affinity of antibody is measured the result
Fig. 2. the electrophorogram of vWF behind the purifying
Fig. 3. the electrophorogram of blood coagulation factor VIII behind the purifying
Embodiment
Embodiment 1, MONOCLONAL ANTIBODIES SPECIFIC FOR
Recombinant human vWF 0.5mg/ml solution mixes with isopyknic Freund's complete adjuvant, processes conventional subcutaneous multi-point injection Balb/c mouse behind the emulsifying agent with emulsor, and every some injection 50ul injects 4 points altogether.Later on whenever at a distance from 3 weeks getting the same dose immunogen and the equal-volume incomplete Freund's adjuvant is processed emulsifying agent; Twice of booster immunization; Three times serum titer is measured in the immunity back, gets the high mouse peritoneal booster immunization of serum titer once, takes out spleen after 4 days; With ordinary method separating spleen cell, and definite cytoactive is greater than 90%.Spleen cell and SP2/0 murine myeloma cell are mixed at 4: 1 centrifugal, get deposition, add PEG (1400MW) at 37 ℃ and carry out cytogamy.In 96 orifice plates, cultivate, add HAT after 24 hours and select nutrient solution, change fresh culture every day, until growing the clone.As envelope antigen, measure cell conditioned medium liquid with recombinant human vWF albumen, choose positive hole and carry out cloning, obtain the hybridoma cell strain of stably excreting vWF monoclonal antibody specific through limiting dilution with the ELISA method.Choose hybridoma cell strain, adopt the known technical scheme of professional domain personnel, utilize culturing bottle or bio-reactor to carry out cell cultures; The collecting cell culture supernatant; Utilize Protein G to carry out affinity purification, packing after evaluation antibody purity and the concentration is in-20 ℃ of cryopreservation.
The acquisition of embodiment 2, monoclonal antibody gene and the structure of expression vector
Collect cultured cells, (Invitrogen) adds TRIzol to specifications, extracts total RNA, electrophoresis detection RNA quality, and UV measures concentration.Be reversed to cDNA by Roche reverse transcription test kit operation instructions ,-20 ℃ frozen subsequent use.Adopt the known technical scheme of professional domain personnel, reference (Ying W et al., BMC Bioinformatics.2006; 7 (Suppl 4): S9)) design primer is the template light chain and the heavy chain fragment of PCR acquisition antibody respectively with cDNA.Light chain and heavy chain fragment TA to the pcDNA3 T carrier that can be used for eukaryotic cell expression, are made up pcDNA3-anti-vWF-L and pcDNA3-anti-vWF-H expression vector.Transformed into escherichia coli, the picking positive colony, order-checking is identified.Analyze sequencing result, obtain correct light chain and heavy chain amino acid sequence.The aminoacid sequence of light chain is seen SEQ ID NO:1, and the aminoacid sequence of heavy chain is seen SEQ ID NO:2.
The mensuration of embodiment 3, antibody affinity costant
With 2ug/ml vWF coated elisa plate, 4 ℃ encapsulate and spend the night.Room temperature was with 2%BSA sealing 1 hour.To resist the vWF antibody dilution to become a series of different concns, concentration is respectively 0.023,0.046,0.092,0.188,0.375,0.75,1.5,3,6ug/ml.To dilute back antibody then and join respectively in the enzyme plate that encapsulates vWF, the room temperature effect is more than 18 hours.Through anti-vWF monoclonal antibody concentration total before the Elisa detection reaction and reaction back remaining anti-vWF monoclonal antibody concentration [F], obtain i.e. [B] value of bonded antibody amount through both difference.Through being the X axle with [B] value, be that the Y axle is drawn (seeing figure .1) with [B]/[F] value then, calculating Kd is 0.06nM.
Embodiment 4, the anti-vWF antibody producing of reorganization
Get HEKC's 293 cells that are in logarithmic phase, the centrifugal cell density that goes down to posterity is 0.5X10 6Cells/ml, every bottle of culture of putting 30ml in the triangular flask of 100ml.Put in the shaking table 100~130rpm, 36.5 ℃, 5%CO 2The following cultivation.Carried out transfection in second day; Get light chain pcDNA3-anti-vWF-L and each 15ug of heavy chain pcDNA3-anti-vWF-H carrier DNA respectively, mixed diluting mixes in the 150mM NaCl solution of 1.5ml; The high efficiency transfection reagent Sinofection (Beijing justice is stuck up Divine Land Bioisystech Co., Ltd) that gets 90ul is diluted in the 150mM NaCl solution of 1.5ml; Mix, then solution in two is mixed, be mixed with the mixed solution of DNA-Sinofection.Hatch 10~20min under the room temperature, dropwise add then and want in the cells transfected suspension-s.Cell after the transfection continues at 100~130rpm, and 36.5 ℃, 5%CO 2Cultivated 5 days in the shaking table.
The harvested cell nutrient solution, centrifugal 10 minutes of 3000g gets supernatant, the 0.45um micro-filtrate membrane filtration.Get the chromatography column that 1ml protein G Sepharose (GE) is housed; With 20mM phosphoric acid salt pH 7.0 damping fluid balance chromatography columns; The supernatant of cell culture fluid to be to flow through chromatography column in 0.5ml/ minute, and the absorbancy that is washed till effluent with 20mM phosphoric acid salt pH 7.0 damping fluids is again reduced to baseline.With 100mM glycocoll pH 3.0 eluant solution antibody, elutriant is neutralized to neutrality with 1M Tris pH 8.0 solution immediately.Gained antibody-20 ℃ preservation.
The preparation of embodiment 5, immobilization monoclonal antibody affinity media
The monoclonal anti body and function desalting column that purifying is good changes damping fluid into 0.1M NaHCO 3, the damping fluid of pH 8.3.The sepharose of cyanogen bromide-activated (GE) is to specifications with 1mM HCl washing 5 times.Get the sepharose that 1ml handles well and mix room temperature reaction 4 hours with the 6mg monoclonal antibody.The 0.1M Tris damping fluid that adds 2ml pH 8.0 continues reaction 2 hours.With the good sepharose of PBS washing coupling, 4 ℃ of preservations are subsequent use.
Embodiment 6, with immobilization monoclonal antibody affinity media purifying vWF
The affinity chromatography medium of the getting embodiment 5 preparation 1ml chromatography column of packing into is with level pad (pH 7.4 for 20mMTris, 500mM NaCl) balance.50ml expresses the cell culture fluid of vWF, and centrifugal 10 minutes of 4000g gets supernatant and uses the 0.45um filtering with microporous membrane.Then with 0.5ml/min flow velocity sample introduction.Sample introduction finishes the back with the unconjugated impurity of level pad flush away, uses elutriant (pH 3.0 for 100mM glycocoll, 10mMNaCl) wash-out then.Elutriant transfers to neutrality with 1M pH 8.0Tris solution.The electrophorogram of vWF behind the purifying is seen Fig. 2.
Embodiment 7, with immobilization monoclonal antibody affinity media purifying blood coagulation Factor IX
The affinity chromatography medium of the getting embodiment 5 preparation 1ml chromatography column of packing into is with level pad (pH 7.4 for 20mMTris, 500mM NaCl) balance.50ml expresses the cell culture fluid of vWF and blood coagulation factor VIII simultaneously, and centrifugal 10 minutes of 4000g gets supernatant and uses the 0.45um filtering with microporous membrane.Then with 0.5ml/min flow velocity sample introduction.Sample introduction finishes the back with the unconjugated impurity of level pad flush away, then with the damping fluid that dissociates (50mM Tris, 250mM CaCl 2, pH 7.3) and the wash-out blood coagulation factor VIII.At last with regeneration damping fluid (pH 3.0 for 100mM glycocoll, 10mM NaCl) the wash-out vWF chromatography media of regenerating.The electrophorogram of the blood coagulation factor VIII behind the purifying is seen Fig. 3.
Figure ISA00000397016200011
Figure ISA00000397016200021
Figure ISA00000397016200031
Figure ISA00000397016200041
Figure ISA00000397016200051
Figure ISA00000397016200061

Claims (10)

1. anti-vWF monoclonal antibody, it is characterized in that: the light chain complementary determining region comprises the CDR1 that sequence is SEQ IDNO:5, and sequence is the CDR2 of SEQ ID NO:6, and sequence is the CDR3 of SEQ ID NO:7; The heavy chain complementary determining region comprises the CDR1 that sequence is SEQ ID NO:8, and sequence is the CDR2 of SEQ ID NO:9, and sequence is the CDR3 of SEQ ID NO:10.
2. the said antibody of claim 1, its light chain variable region amino acid sequence is SEQ ID NO:3; The weight chain variable region amino acid sequence is SEQ ID NO:4.
3. the said antibody of claim 1, its light-chain amino acid sequence is SEQ ID NO:1; Heavy chain amino acid sequence is SEQ ID NO:2.
4. the said antibody of claim 1 is the monoclonal antibody that mouse monoclonal antibody or other modes change structure.
5. the said antibody of claim 1 is the monoclonal antibody through hybridoma or recombination cell preparation.
6. method of utilizing the said antibody purification vWF of claim 1, it is characterized in that: (1) will resist vWF antibody to be fixed on the solid-phase matrix, process affine sorbing material; (2) with the vWF in the affinitive material absorption material solution of processing; (3) with elution buffer wash-out vWF.
7. the method for the said purifying vWF of claim 6 is characterized in that elution buffer is the acidic buffer of pH2~pH3.
8. method of utilizing the said antibody purification blood coagulation factor VIII of claim 1, it is characterized in that: (1) will resist vWF antibody to be fixed on the solid-phase matrix, process affine sorbing material; (2) with the vWF-FVIII complex body in the affinitive material absorption material solution of processing; (3) with the buffer solution elution blood coagulation factor VIII that dissociates; (4) with regeneration buffer solution elution vWF.
9. the method for the said purifying blood coagulation Factor IX of claim 8, the damping fluid that it is characterized in that dissociating is the neutral buffered liquid that contains 200~300mM CaCl2 pH 6~8.
10. the method for the said purifying blood coagulation Factor IX of claim 8, the damping fluid that it is characterized in that regenerating is the acidic buffer of pH2~pH3.
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Cited By (7)

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Publication number Priority date Publication date Assignee Title
CN103351431A (en) * 2012-11-13 2013-10-16 蔡胜和 Coagulation factor 8 and its mutant purifying method
CN103760358A (en) * 2013-11-12 2014-04-30 赵铁铭 Reagent for detecting Von Willebrand factor and preparation method and application thereof
WO2014075205A1 (en) * 2012-11-15 2014-05-22 Cai Shenghe Method for purifying blood coagulation factor viii and the variants thereof
CN105400769A (en) * 2015-11-17 2016-03-16 清华大学 Hybridoma cell strain for anticoagulation factor VIII monoclonal antibody
CN108997501A (en) * 2018-09-01 2018-12-14 无锡傲锐东源生物科技有限公司 Anti- VWF protein monoclonal antibody and application thereof
CN117069835A (en) * 2023-06-05 2023-11-17 北京大学第一医院 A kind of monoclonal antibody against vWF/PF4 protein and its application
CN117192132A (en) * 2023-11-03 2023-12-08 庄亚(北京)生物科技有限公司 vWF fragment residue detection kit and method

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CN1174575A (en) * 1994-11-30 1998-02-25 味之素株式会社 Antithrombotic agent and anti-von willebrand factor monoclonal antibodies
CN1311691A (en) * 1998-08-19 2001-09-05 味之素株式会社 Antithrombotic agent and humanized anti-von willebrand factor monoclonal antibody

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CN1174575A (en) * 1994-11-30 1998-02-25 味之素株式会社 Antithrombotic agent and anti-von willebrand factor monoclonal antibodies
CN1311691A (en) * 1998-08-19 2001-09-05 味之素株式会社 Antithrombotic agent and humanized anti-von willebrand factor monoclonal antibody

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103351431A (en) * 2012-11-13 2013-10-16 蔡胜和 Coagulation factor 8 and its mutant purifying method
WO2014075205A1 (en) * 2012-11-15 2014-05-22 Cai Shenghe Method for purifying blood coagulation factor viii and the variants thereof
CN103760358A (en) * 2013-11-12 2014-04-30 赵铁铭 Reagent for detecting Von Willebrand factor and preparation method and application thereof
CN105400769A (en) * 2015-11-17 2016-03-16 清华大学 Hybridoma cell strain for anticoagulation factor VIII monoclonal antibody
CN105400769B (en) * 2015-11-17 2019-04-12 清华大学 The hybridoma cell strain of anticoagulin VIII monoclonal antibody
CN108997501A (en) * 2018-09-01 2018-12-14 无锡傲锐东源生物科技有限公司 Anti- VWF protein monoclonal antibody and application thereof
CN117069835A (en) * 2023-06-05 2023-11-17 北京大学第一医院 A kind of monoclonal antibody against vWF/PF4 protein and its application
CN117069835B (en) * 2023-06-05 2024-01-30 北京大学第一医院 anti-vWF/PF 4 protein monoclonal antibody and application thereof
CN117192132A (en) * 2023-11-03 2023-12-08 庄亚(北京)生物科技有限公司 vWF fragment residue detection kit and method
CN117192132B (en) * 2023-11-03 2024-02-02 庄亚(北京)生物科技有限公司 vWF fragment residue detection kit and method

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