CN102010472A - Anti-D-dimer monoclonal antibody and application thereof - Google Patents
Anti-D-dimer monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-D-dimer monoclonal antibody. The amino acid residue sequence of the light chain of the antibody is shown in SEQ ID No.1, and the amino acid residue sequence of the heavy chain of the antibody is shown in SEQ ID No.2; and the antibody is secreted by a hybridoma cell strain 6B8D11H12 the preservation number of which is CCTCC NO.C201060. The anti-D-dimer monoclonal antibody can be used in preparing various D-dimer aspartame assay kits. By utilizing the anti-D-dimer monoclonal antibody disclosed by the invention and a D-dimer aspartame assay kit prepared by gold immunochromatographic assay(GICA), the detection sensitivity is up to 96.08%, the detection specificity is up to 94.79%, the detection accuracy is up to 95.67%, the Kappa detection value is up to 0.901 which is greater than 0.75, and the diagnosis is more consistent with clinical diagnosis, thus the anti-D-dimer monoclonal antibody can be used for aided diagnosis on thrombotic diseases in clinical applications.
Description
Technical field
The present invention relates to a kind of anti-D-dimer monoclonal antibody and uses thereof, belong to biological technical field.
Background technology
The D-dimer is the dead end product of crosslinked fibrin after the plasmin effect.In coagulation process, zymoplasm is after hydrolysis of fibrin is former, promptly discharge fibrinopeptide A (FPA) and peptide B (FPB) in succession, remainder is SFM (SFM), under the Ntn hydrolase effect of changing one's profession, SFM changes scleroproein into, then blood coagulation, its process is to finish after crosslinked through a series of, and after this scleroproein character of Xing Chenging is stablized, and does not generally dissolve, but can be degraded by plasmin, crosslinked fibrin generates some kinds of polymers gradually in the plasmin degradation process, the D-dimer promptly is one of its special product, and its molecular weight is 184000～202000.Under pathological state, the running balance of blood coagulation and fibrinolytic is destroyed, and the blood coagulation tendency strengthens, thereby fibrin degradation product (FDP) increases, and causes the D-dimer content to increase.Increasing of D-Dimer levels, showing has fibrinous thrombus to form and the fibrinolytic generation in the body, so can be used as the molecular marker of interior hypercoagulative state of body and hyperfibrinolysis clinically.
The dimeric detection of D-has significant values to the diagnosis and the treatment of multiple disease.Especially can in asymptomatic high-risk patient, carry out early screening to deep venous thrombosis (DVT); Can be used as the first-selected shaker test of getting rid of the diagnosis pulmonary infarction with PAO2-Pao2 (A-aDO2) joint-detection.But auxiliary diagnosis hepatic diseases in addition; Cerebral infarction is diagnosed and the prognosis judgement; Differentiate malignant tumour, other factors that can cause that the D-dimer raises also have: traumatic fracture, severe infections, SCHD, severe infection, children's anaphylactoid purpura (HSP) acute phase, pyemia, sarcoidosis etc.
At present, the dimeric detection method of D-is mainly contained: 1) ELISA method: Rylatt at first with the D-dimer of purifying as antigen, obtain the dimeric monoclonal antibody of the anti-D-of many strains behind the immune mouse, use wherein D-dimer content of ELISA test determination then, its sensitivity reaches 10ng/ml, is called the ELISA detection method.Its susceptibility height can be quantitative, but complex operation.2) latex agglutination method: be present most widely used D-dimer detection method.By on latex particle (Latex), when antigen antibody reaction took place for D-dimer and Latex in the blood, the Latex of sensitization generation aggegation recorded its content, is the Latex detection method with the monoclonal antibody bag that obtains.This law tolerance range is low, can only be qualitative, be adapted to emergency treatment.3) chromophoric substrate method: this method susceptibility is good, accuracy is high, can detect rapidly by single part of sample, but cost is too high.
Adopt the immunochromatographic method of colloidal gold-labeled method to have convenient, fast, reagent and be easy to advantages such as preservation, be widely used in the every field of immunity, but the use key of colloidal gold immunity chromatography will solve the problem of its specificity, sensitivity and accuracy, and this all needs to select for use the monoclonal antibody with high degree of specificity and high-affinity.
Summary of the invention
The purpose of this invention is to provide a kind of anti-D-dimer monoclonal antibody and the application in the D-dimer detection kit of colloidal gold immunity chromatography preparation thereof of high degree of specificity, with realize utilizing colloidal gold immunity chromatography can be fast, accurately, the dimeric purpose of high-sensitivity detection D-.
Anti-D-dimer monoclonal antibody provided by the present invention is characterized in that: the amino acid residue sequence of its light chain is shown in SEQ ID No.1, and the amino acid residue sequence of its heavy chain is shown in SEQ ID No.2.
Described anti-D-dimer monoclonal antibody is to be by preserving number: the hybridoma cell strain 6B8D11H12 secretion of CCTCC NO.C201060 produces.
Described hybridoma cell strain is to be immunogenic with the D-dimer, and mouse is immune object, by the immunization mouse, gets the immune mouse spleen and prepares suspension, and make it to carry out cytogamy with the myeloma cell and obtain.
Described anti-D-dimer monoclonal antibody belongs to the IgG hypotype.
It is 1: 10 that the ascites of described anti-D-dimer monoclonal antibody is tired
6
Anti-D-dimer monoclonal antibody of the present invention not only can combine with D-dimer high degree of specificity, and highly sensitive, good stability, can be prepared into the dimeric various detection kit of D-by method known in those skilled in the art.Especially, use the D-dimer detection kit of anti-D-dimer monoclonal antibody of the present invention and colloidal gold immunity chromatography preparation, not only can quick, simple and direct detection human serum, the D-dimer in blood plasma or the whole blood sample, not only make detection sensitivity reach 96.08%, detect specific degree and reach 94.79%, accuracy in detection reaches 95.67%, and can make the Kappa test value reach 0.901, much larger than 0.75, have higher diagnosis consistence with clinical diagnosis, can be used for clinically auxiliary diagnosis thrombotic diseases.
Description of drawings
Fig. 1 is 5 ' RACE product electrophorogram, and wherein No. 1 is the light chain result, No. 2 chain results that attach most importance to, and M is DNA Marker.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
The experimental technique of unreceipted actual conditions in the following example, the condition of advising according to manufacturer usually according to the conditioned disjunction described in normal condition, the laboratory manual.
Embodiment 1: anti-D-dimer MONOCLONAL ANTIBODIES SPECIFIC FOR
One, animal immune
Get the BALB/c mouse in female 6～8 ages in week, first immunisation concentration is the pure product of D-dimer of 5mg/ml, through abdominal cavity and the injection of four limbs oxter, total amount 1ml; Every 2 weeks with same method booster immunization 1 time, immunity is 3 times altogether, after the last immunity the 4th day, from through three immunity back eyeball of mouse blood samplings, centrifugation serum is selected the mouse preparation fusion of tiring high with the ELISA method.
Two, the preparation of hybridoma cell line
Will finish the mouse of immunologic process prepare extracting spleen cell and merge with murine myeloma cell SP2/0, prepare feeder cell the day before yesterday in fusion; Get mouse boosting cell under aseptic during fusion, mixed with 10: 1,, be resuspended in the HAT selective medium after centrifugal, be inoculated in the 96 hole microwell plates that contain feeder cell, put 37 ℃, 5%CO with the 50%PEG mediates fusion with the SP2/0 cell
2Incubator is cultivated, and 3d, 5d, 7d change liquid with HAT nutrient solution half amount after merging, and use the HT nutrient solution after two weeks instead.
Observe the growing state of hybridoma, wait the clone to grow to 1/3～1/2 of hole floorage, get culture supernatant, carry out antibody test, screening positive clone with the ELISA method.With limiting dilution assay the hybridoma in positive hole being carried out cloning and cultivate, is 100% up to cloning cell antibody positive rate, and selecting the strain of high secretion specific cell (is that ELISA tires at 1: 10
6Above positive colony), this moment can be with the further enlarged culturing of positive colony cell.Hybridoma more than 3 months and frozen repeatedly, recovery, is regularly collected supernatant through external continuous passage, measure antibody in the supernatant with the method for screening antibody, can the stably excreting monoclonal antibody until clone.
Three, MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
Select adult BALB/c mouse for use, abdominal injection 0.5ml whiteruss, good this cell strain of monoclonal hybridoma strain 6B8D11H12[Chinese typical culture collection center (CCTCC) preservation in being arranged at Chinese Wuhan University of 1～2 week back collection growth conditions, preservation date is: on July 1st, 2010, preserving number is: CCTCC NO.C201060], every abdominal injection 1ml.As seen the mouse web portion of inoculating about 2 weeks obviously expands, and opens the abdominal cavity after putting to death with the cervical vertebra dislocation method, takes out ascites.Supernatant is drawn in centrifugal back, behind ammonium sulfate precipitation, uses DEAE ion-exchange column purification again, with the acetate buffer wash-out of PH5.6,20mM, collects elutriant.Use affinitive layer purification method (albumin A-Sepharose4B post) to continue purifying, last batten spare again: sample and albumin A-Sepharose4B post is gone up sample again with after the Tris damping fluid balance of PH8.2,1.0M.Elution requirement: with the glycine buffer wash-out of PH3.0,50mM, collect elutriant, make special monoclonal antibody.
Four, the ascites of monoclonal antibody is tired and is identified
Get mouse ascites antibody, tiring with the method mensuration of screening antibody is 1: 10
6, hypotype is accredited as the IgG type.
Embodiment 2: the specificity of monoclonal antibody is identified
Material: Fibrinogen and fragment X, Y, D, E and blood plasma.
Method: adopt the ELISA method to detect, with the positive contrast of D-dimer.
The result: display fibers proteinogen and fragment X, Y, D, E and blood plasma are all negative.
Illustrate: anti-D-dimer monoclonal antibody of the present invention has high degree of specificity to the D-dimer.
Embodiment 3: the clone of the variable region sequences of monoclonal antibody and order-checking
Adopt 5 ' RACE (Rapid Amplification of cDNA Ends, rapid amplifying cDNA end) technology, the variable region sequences of cloning function antibody from the hybridoma cell strain 6B8D11H12 that secretes anti-D-dimer monoclonal antibody.Its step can be sketched and be: the gene-specific primer (GSP1) by an antisense synthesizes cDNA first chain, behind the cDNA first chain purifying, (Terminal deoxynucleotidy transferase, TdT) 3 ' end at cDNA adds a synthetic homopolynucleotide anchor series to utilize terminal deoxynucleotidyl transferase.Utilize second nido gene-specific primer (GSP2) and one and the homopolynucleotide tail cDNA that can the annealed anchor primer increases.Concrete experimentation is as follows:
Material:
-anti-D-dimer the monoclonal antibody that produces by hybridoma cell strain 6B8D11H12 secretion
-intestinal bacteria Top10 bacterial strain
-pGEM-T Easy cloning vector
Design of primers:
According to the characteristics of immunoglobulin gene, gene-specific primer GSP1, the GSP2 of design two cover corresponding Ig of difference and Kappa constant region, primer sequence is as follows:
pRace-H-GSP1：GTCCACCKYGGTSYTGCTGGCYGGGTG
pRace-H-GSP2：GCACACYRCTGGACAGGGATCCAGAGTTCC
pRace-K-GSP1：ACTTGACATTGATGTCTTTG
pRace-K-GSP2：CACGACTGAGGCACCTCCAGATG
Clone's process:
A, first chain synthesize
With total RNA is template, is primer with GSP1, synthetic cDNA first chain under the effect of ThermoScript II.Concrete steps are as follows:
1) in the Eppendorf tube of a 0.5ml, add following component:
GSP1 2.5ml
Replenishing water to the cumulative volume of handling through DEPC is 15.5 μ l, mixing.
2) 70 ℃ of sex change 10min, ice bath 1min of short durationly adds following component after centrifugal successively:
10*PCR?buffer 2.5μl
25mM?MgCl
2 2.5μl
10mM?dNTP?mix 1.0μl
0.1M?DTT 2.5μl。
3) mixing, of short duration centrifugal rearmounted 42 ℃ of 1min.
4) in reaction system, add 1 μ l SuperScriptTMII RT (Invitrogen company), gently the rearmounted 42 ℃ of reaction 50min of mixing.
5) reverse transcription finishes 70 ℃ of postposition, 15min with termination reaction.
6) be placed on 37 ℃ in centrifugal 10～20 seconds.
7) add 1 μ l RNase mix, mixing is placed on 37 ℃ of reaction 30min gently.
8) put the reaction tubes taking-up standby on ice.
B, DNA purifying
Use the QIAGEN QIAquick PCR of company purification kit.
1) add 5 times of volumes in the PB of reverse transcription system damping fluid in the reverse transcription reaction pipe, mixing.
2) the centrifugal post of QIAquick is placed the 2ml collection tube, sample is added centrifugal post, centrifugal 60 seconds of 13000rpm.
3) discard liquid, centrifugal post is put back in the original collection tube, add 0.75ml PE damping fluid in the centrifugal post of QIAquick, centrifugal 60 seconds of 13000rpm.
4) discard liquid, the centrifugal post of QIAquick is put back to original collection tube, centrifugal 60 seconds of 13000rpm.
5) the centrifugal post of QIAquick is placed a clean 1.5ml centrifuge tube, after QIAquick film central authorities add 30 μ l EB damping fluids, leave standstill 1min, centrifugal 60 seconds of 13000rpm.
C, cDNA tailing
1) in the Eppendorf tube of a 0.5ml, once add following component:
The water 6.5 μ l that DEPC handles
5*tailing?buffer 5.0μl
2mM?dCTP 2.5μl
The cDNA 10.0 μ l of purifying, mixing.
2) reaction tubes is put 94 ℃ keep 3min after, ice bath 1min, of short duration centrifugal postposition on ice.
3) add 1 μ l TdT, the rearmounted 37 ℃ of reaction 10min of mixing.
4) reaction tubes is placed 65 ℃ of effect 30min termination reactions, centrifugal be placed on standby on ice.
D、PCR
In the PCR of 0.2ml thin-walled tube, add following component:
Aqua sterilisa 31.5 μ l
10*PCR?Buffer 5.0μl
25mM?MgCl
2 3.0μl
10mM?dNTP?mix 1.0μl
GSP2(10pmol/μl)?2.0μl
AAP(10pmol/μl) 2.0μl
dC-tailed?cDNA 5.0μl
Tag enzyme 0.5 μ l
Add up to 50.0 μ l
React in the rearmounted PCR instrument of mixing.
E, PCR product electrophoresis purifying
The PCR product identifies through 1.5% agarose gel electrophoresis, the result as shown in Figure 1: wherein No. 1 is the light chain result, at about 450bp place one specific band is arranged; The chain result that attaches most importance to for No. 2 has a specific band at about 500bp place.
The pGEM-T Easy test kit of F, use Promega company, to the pGEM-TEasy carrier, transformed into escherichia coli Top10 (Invitrogen company) detailed process is with the PCR product cloning:
1) light chain and heavy chain PCR product reclaim the band of 450bp and 500bp respectively through 1.5% agarose gel electrophoresis, and final constant volume is in the aqua sterilisa of 20 μ l volumes;
2) in the Eppendorf tube of a 0.5ml, add following component:
2* connects damping fluid 5 μ l
pGEM-T?Easy 50ng(1μl)
PCR product 25ng (3 μ l)
The room temperature connection connected more than 16 hours in 2 hours or 4 ℃ behind the mixing.
3) transformed into escherichia coli competent cell is coated on the LB flat board that contains penbritin and IPTG, X-GAL, cultivates about 12 hours for 37 ℃.
The base sequence of the PCR product that G, mensuration are cloned
Respectively choose at least 5 plasmids and measure the base sequence of its contained PCR product.
H, according to the corresponding relation of base sequence and amino acid coding, analyze the reading frame of PCR product, determine corresponding amino acid sequences, the sequence of its light chain and heavy chain is respectively shown in SEQ ID No.1 and SEQ ID No.2.Verify that according to the characteristics of immunoglobulin gene it is an antibody sequence.
Embodiment 4: the application of anti-D-dimer monoclonal antibody of the present invention
Adopt colloidal gold immunity chromatography known in those skilled in the art, make D-dimer quick detection kit, the D-dimer in external qualitative detection human serum, blood plasma or whole blood sample is used for auxiliary diagnosis thrombotic diseases clinically.
One, detects principle
Adopt the immunochromatography technique of colloid gold label, D-dimer in the sample combines with the anti-D-dimer monoclonal antibody of detection zone bag quilt, when D-dimer concentration surpasses detectability, can form a colour band at detection zone, the dimeric sample of no D-can not form colour band at detection zone; The Quality Control district then red ribbon can occur all the time, and is correctly effective with the prompting detected result.
Two, detection method
A, specimen collection and preparation
1) adopt the standard laboratory program to collect serum, blood plasma or whole blood sample.
2) avoid hot deactivation sample, to prevent haemolysis or protein denaturation.
3) whole blood sample collection: use heparin or EDTA to be antithrombotics, collect blood preparation, because the D-dimer is very unstable in whole blood or serum specimen, whole blood or serum specimen should be used for detecting in back 4 hours in collection.
4) plasma collection of specimens: whole blood sample is centrifugal to obtain serum or plasma specimen, if sample can not be used for detection at once, put 2～8 ℃ and preserved 24 hours, if can not in 24 hours, detect, need put-20 ℃ or more low temperature preservation down.[note: sample needs balance to room temperature before detection.】
B, operation steps
1) before the experiment with desired substance and sample balance to room temperature.Tear aluminium foil bag then, therefrom take out check-out console, be placed on the surface of level.
2) add 80 μ l samples (or with suction pipe drip 2 samples) in sample well with pipettor.
3) observations in 15 minutes.[note: if the low sample of D-dimer content, developing time may be above 15 minutes.】
Three, the result judges
1) positive: when two colour bands occurring in 15 minutes, the result is judged to the positive.
2) feminine gender: if colour band does not appear in detection zone, and a colour band occurs in the Quality Control district, the result is judged to feminine gender.
3) invalid: if colour band does not appear in the Quality Control district, the result is judged to invalid, must use new check-out console to detect sample again.
Four, clinical application result
For estimating suitability and the accuracy that D-dimer quick detection kit of the present invention is applied to thrombotic diseases is carried out auxiliary diagnosis clinically, carried out clinical study in Shanghai City Hospital of Combination of Chinese Traditional and Western Medicin, from clinical, selected 300 routine samples as research object.Selected object also comprises the prescription on individual diagnosis patient of the non-thrombotic diseases of small part based on the patient of thrombotic diseases.Adopt the D-dimer detection kit (immunofluorescence technique) of American I nverness company to compare research, sample is divided into case group and control group according to detected result.With test kit of the present invention these samples are detected simultaneously, compare detected result, and carry out statistical study, experimental result is shown in Table 1.
Table 1 is used the Clinical Comparison Study result of detection kit of the present invention
The result shows by table 1: the detection sensitivity of test kit of the present invention reaches 96.08%, detects specific degree and reaches 94.79%, and accuracy reaches 95.67%, can be applicable to clinical thrombotic diseases is carried out auxiliary diagnosis.
In addition, the Kappa test value is 0.901, much larger than 0.75, illustrates that test kit of the present invention and clinical diagnosis have higher diagnosis consistence.
Claims (8)
1. anti-D-dimer monoclonal antibody, it is characterized in that: the amino acid residue sequence of its light chain is shown in SEQ ID No.1, and the amino acid residue sequence of its heavy chain is shown in SEQ ID No.2.
2. anti-D-dimer monoclonal antibody according to claim 1 is characterized in that: described anti-D-dimer monoclonal antibody is to be by preserving number: the hybridoma cell strain 6B8D11H12 secretion of CCTCC NO.C201060 produces.
3. anti-D-dimer monoclonal antibody according to claim 2, it is characterized in that: described hybridoma cell strain is to be immunogenic with the D-dimer, mouse is immune object, by the immunization mouse, get the immune mouse spleen and prepare suspension, and make it to carry out the cytogamy acquisition with the myeloma cell.
4. anti-D-dimer monoclonal antibody according to claim 1 is characterized in that: described anti-D-dimer monoclonal antibody belongs to the IgG hypotype.
5. anti-D-dimer monoclonal antibody according to claim 1 is characterized in that: it is 1: 10 that the ascites of described anti-D-dimer monoclonal antibody is tired
6
6. the application of the described anti-D-dimer monoclonal antibody of claim 1 is characterized in that: be used for the preparation of D-dimer detection kit.
7. the application of anti-D-dimer monoclonal antibody according to claim 6 is characterized in that: the D-dimer detection kit that is used for the colloidal gold immunity chromatography preparation.
8. the application of anti-D-dimer monoclonal antibody according to claim 7 is characterized in that: be used for clinically the auxiliary diagnosis to thrombotic diseases.
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| CN102692504A (en) * | 2012-06-26 | 2012-09-26 | 南京基蛋生物科技有限公司 | D-dimer quantitative fluorescence immunoassay test strip and preparation method thereof |
| CN103468644A (en) * | 2013-09-26 | 2013-12-25 | 重庆探生科技有限公司 | Hybridoma cell line capable of generating monoclonal antibody for resisting human D-dimer, preparation method and application |
| CN104730245A (en) * | 2014-11-28 | 2015-06-24 | 威海纽普生物技术有限公司 | D-dimer detection kit and D-dimer detection method |
| CN105044331A (en) * | 2015-06-01 | 2015-11-11 | 上海凯创生物技术有限公司 | Detection kit with D-dimer colloidal gold method |
| CN105891505A (en) * | 2015-03-31 | 2016-08-24 | 北京科美生物技术有限公司 | Colloidal gold immunocolorimetry kit for detecting D-dimer (DD) and preparation method of kit |
| CN109053893A (en) * | 2018-08-15 | 2018-12-21 | 苏州智维安生物技术有限公司 | A kind of anti-D-dimer antibody and preparation method thereof |
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| CN102692504A (en) * | 2012-06-26 | 2012-09-26 | 南京基蛋生物科技有限公司 | D-dimer quantitative fluorescence immunoassay test strip and preparation method thereof |
| CN103468644A (en) * | 2013-09-26 | 2013-12-25 | 重庆探生科技有限公司 | Hybridoma cell line capable of generating monoclonal antibody for resisting human D-dimer, preparation method and application |
| CN103468644B (en) * | 2013-09-26 | 2016-01-20 | 重庆探生科技有限公司 | Produce hybridoma cell strain and the preparation method and application of the monoclonal antibody of anti-human DDi |
| CN104730245A (en) * | 2014-11-28 | 2015-06-24 | 威海纽普生物技术有限公司 | D-dimer detection kit and D-dimer detection method |
| CN105891505A (en) * | 2015-03-31 | 2016-08-24 | 北京科美生物技术有限公司 | Colloidal gold immunocolorimetry kit for detecting D-dimer (DD) and preparation method of kit |
| CN105044331A (en) * | 2015-06-01 | 2015-11-11 | 上海凯创生物技术有限公司 | Detection kit with D-dimer colloidal gold method |
| CN109053893A (en) * | 2018-08-15 | 2018-12-21 | 苏州智维安生物技术有限公司 | A kind of anti-D-dimer antibody and preparation method thereof |
| CN111647085A (en) * | 2020-08-04 | 2020-09-11 | 苏州方德门达新药开发有限公司 | Antibodies, methods of making and uses thereof |
| CN113717285A (en) * | 2021-11-04 | 2021-11-30 | 北京沃森赛瑟生物技术有限公司 | Anti-human D-dimer antibodies and uses thereof |
| CN113717285B (en) * | 2021-11-04 | 2022-02-01 | 北京沃森赛瑟生物技术有限公司 | Anti-human D-dimer antibodies and uses thereof |
| CN116143917A (en) * | 2021-11-20 | 2023-05-23 | 东莞市朋志生物科技有限公司 | anti-D-dimer antibody and preparation method and application thereof |
| WO2023088445A1 (en) * | 2021-11-20 | 2023-05-25 | 东莞市朋志生物科技有限公司 | Anti-d-dimer antibody, and preparation method therefor and use thereof |
| CN116143917B (en) * | 2021-11-20 | 2023-10-31 | 东莞市朋志生物科技有限公司 | anti-D-dimer antibody and preparation method and application thereof |
| CN115948342A (en) * | 2022-12-19 | 2023-04-11 | 山东大学 | CHO cell strain, D-dimer monoclonal antibody and application thereof |
| CN115948342B (en) * | 2022-12-19 | 2023-10-03 | 山东大学 | CHO cell strain, D-dimer monoclonal antibody and application thereof |
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| CN102010472B (en) | 2012-09-05 |
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