CN105754953A - Monoclonal antibodies resistant to PLAG (platelet-aggregation-stimulating) domain of human PDPN (Podoplanin) and application of monoclonal antibodies - Google Patents
Monoclonal antibodies resistant to PLAG (platelet-aggregation-stimulating) domain of human PDPN (Podoplanin) and application of monoclonal antibodies Download PDFInfo
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Abstract
The invention discloses monoclonal antibodies resistant to a PLAG (platelet-aggregation-stimulating) domain of human PDPN (Podoplanin) and an application of the monoclonal antibodies. The monoclonal antibodies are secreted by hybridoma cell lines with preservation numbers of CCTCC NO: C201613 and CCTCC NO: C2015185 respectively, are named as SZ-163 and SZ-168 and are both IgG1 subclass antibodies; the monoclonal antibodies are specifically bound with reductive PDPN, are bound with an NCI-H226 cell Lysis solution to generate a binding zone when the molecular weight is 36 kDa, and are bound with a recombinant human PDPN-Fc to generate a binding zone when the molecular weight is 67 kDa. One anti-PDPN monoclonal antibody can block platelet activating aggregation induced through binding of tumor cells and platelets, and the other anti-PDPN monoclonal antibody can be bound with the first functional inhibiting antibody at different epitopes of PDPN. The antibodies can be used for preparing an anti-tumor-metastasis drug or establishing a double-antibody sandwich method for measuring the content of soluble PDPN in human plasma.
Description
Technical field
The present invention relates to anti-human flatfoot albumen (Podoplanin, PDPN) stimulating platelet aggregated structure territory (PLatelet
AGgregation-stimulating, PLAG) monoclonal antibody SZ-163 of functional areas and SZ-168 and preparation method thereof；This
The bright application further to prepared functional antibodies in the medicine of preparation suppression tumor growth and transfer；The present invention relates to list
Clonal antibody application in setting up double-antibody sandwich elisa detection human plasma solubility PDPN.
Background technology
Due to environment, diet, habits and customs and operating pressure, and the factor such as heredity, the incidence of tumour and the death rate one
Straight high, tumour has become the common disease of serious harm human health, frequently-occurring disease.Metastases is that tumor patient is dead
One main cause.Although the mechanism of metastases obtains the attention of clinician and scientific worker for a long time, so far for tumour
The molecular mechanism of transfer is the most not clear.Therefore the transfer for how prevention and control tumour still lacks effective means, in advance
Anti-and suppression metastases will be the emphasis in treatment and prevention of tumour, focus and difficult point within considerably long period.In recent years, increasingly
Many evidence display blood platelets play an important role in metastases.Blood platelet mechanism in metastases may have following
Several: 1. to it is found that (release is the direct induced platelet aggregation of a lot of tumour cell: Tumor cell-induced platelet
Aggregation, TIPA).The a lot of cell factor of intra platelet free calcium of activation, including platelet-derived angiogenesis factor etc..
These cell factors can promote transfer and the growth of tumour cell, contributes to the regeneration of blood vessel；2. tumour cell causes blood platelet
Activate and finally result in integrin glycoprotein-b/-a (GP II b/ III a) by release ADP synthesis thromboxane A2 (TXA2) etc.
Activation.The blood platelet of tumor patient reactivity raises, and more promotes said process and develops；3. the blood platelet of activation is straight
Connect and stick to tumor cell surface, thus help tumour cell escape body immune attack, also contribute to simultaneously tumour cell
Bed, adhere to, thus the transfer of beneficially tumour cell.Finding in the treatment of tumour in recent years, medicament for resisting platelet aggregation should
Treatment for tumor patient, although along with hemorrhage risk, but generation development and the transfer of tumour can be suppressed, therefore
Perhaps can be the generation of tumour to the research of medicament for resisting platelet aggregation mechanism of action in tumor development, growth, turn
Move the Research Thinking new with treatment offer and method.
Now there are some researches show: one of molecule played a crucial role in blood platelet interacts with tumour cell is flatfoot albumen
(Podoplanin, PDPN), PDPN is that I type saliva sticks albumen, and being originally found is at lymphatic endothelial, in being also lymphatic vessel
The Specific marker of skin, at surface expressions such as normal structure such as kidney, type I alveolar cells.The structure of PDPN has three part groups
Becoming, extracellular region is made up of 162 amino acid, containing substantial amounts of serine, threonine residues, EDxxVTPG sheet therein
Section is the domain (platelet aggregation-stimulating, PLAG) that stimulating platelet is assembled, and is positioned at amino acid sequence
29-54 amino acid between, this sequence high conservative between species, PLAG district is made up of three repetitive sequences, main
That to be worked is PLAG3, and its Thr52 is as the site of O-glycan sialylation, and cross-film district is single transmembrane, and intracellular region is
It is made up of 9 amino acid, there is PKA/C phosphorylation site；The CLEC-2 that platelet surface is expressed is the interior of PDPN
Exogenous receptor, is combined with PDPN and depends on that O-glycan is sialic exists center, be independent of plasma fraction, in conjunction with after can be straight
The activation connecing induced platelet is assembled, and is combined weak and not induced platelet aggregation, induced platelet activation populated with other sites
One process postponed.Find a lot of tumour cells (lung neoplasm, stomach neoplasm, intestinal tumor, dermoid cancer, evil recently
Property celiothelioma, Kaposi sarcoma, angiosarcoma, seminoma of testis and brain tumor etc.) also express PDPN, and PDPN
As new tumor markers.Tumor tissues surface expression PDPN raises can cause platelet activation, and this is probably blood platelet and promotees
Enter the transfer of tumour and the basis of growth.Therefore, the antibody of anti-PDPN may stop transfer and the growth of tumour, for tumour medicine
The research and development of thing provide new direction.
The PLAG district function inhibitio monoclonal antibody of anti-PDPN, by suppressing the combination of PDPN Yu CLEC-2, stops blood little
The activation of plate is assembled, and this is not the main path that under physiological status, platelet activation is assembled, so anti-PDPN antibody can not only press down
The transfer of tumour processed, and do not have the hemorrhage risk that medicament for resisting platelet aggregation causes, there is the biggest using value.
Therefore, develop and develop the monoclonal antibody in the PLAG district blocking PDPN and the interphase interaction of CLEC-2, no
Being only capable of stoping tumour cell and hematoblastic combination, thus stop the transfer of tumour, the treatment for tumour provides new direction and side
Method；The monoclonal antibody of the different epitopes for PDPN developed is for double-antibodies sandwich ELISA, in detection cancer
The plasma soluble PDPN level of the patients such as disease provides a feasible method.The level determination of plasma soluble PDPN can conduct
A kind of new tumor markers occurs as tumour and the diagnosis of transfer and prognostic monitoring and differentiation.
Summary of the invention
It is an object of the present invention to provide monoclonal antibody and the application thereof of anti-human flatfoot proteinemina platelet accumulation regions, it is provided that two strains resist
In the functional monoclonal antibody of PDPN, a strain can block the hematoblastic work that tumour cell is induced with hematoblastic combination
Changing and assemble, another strain anti-PDPN monoclonal antibody can be incorporated into the not synantigen of PDPN with the first strain function inhibitio antibody
Epi-position.
It is a further object of the present invention to provide the hybridization of the monoclonal antibody in the PLAG district that can produce above-mentioned specific anti-PDPN
Oncocyte system.
Another object of the present invention be to provide the monoclonal antibody in the PLAG district of above-mentioned specific anti-PDPN prepare antitumor
Application in diversion medicaments and the application in prepared by double-antibodies sandwich ELISA.
The preservation information of cell line is: preservation address: Wuhan University's preservation center, Wuchang District, Wuhan City, Hubei Province, depositary institution:
Wuhan University's China typical culture collection center preservation date: on January 20th, 2016, hybridoma cell strain SZ-163, protects
Hide and number: CCTCC NO:C201613；Hybridoma cell strain SZ-168, deposit number: CCTCC NO:C2015185.
For reaching above-mentioned purpose, the concrete technical scheme of the present invention is, two kinds of hybridoma cell strains, its preparation method includes following
Step:
(1) in order to increase immunogenicity, we use the polypeptide fragment of 22 peptides in the PLAG district of Prof. Du Yucang people PDPN
(DTETTGLEGGVAMPGAEDDVVC) (reality is seen with keyhole limpet hemocyanin (KLH) coupling as immunogene
Execute example 1), immunity Balb/c mouse in conventional manner；
(2) fused cell growth clone is obtained: from aseptic its splenocyte B cell as antigen sensibilization that takes of immuno-competent mouse, routinely
Method, merges B cell with myeloma cell's SP2/0 strain, then utilizes the fused cell HAT screening technique of routine to sieve
Choosing, and then obtain fused cell growth clone；
(3), after applying the immunological technique screenings such as ELISA method, flow cytometry and western blot method and identifying, two strains tools are picked out
There is hybridoma cell strain SZ-163 and SZ-168 of high antibody-secreting level.(seeing embodiment 2)；
In technique scheme, using Prof. Du Yucang 22 peptide-KLH fragment as immunogene, conventional three immunity inoculation 8 week old are female
Balb/c mouse (every minor tick 4 weeks)；With anti-PDPN in enzyme linked immunosorbent assay (ELISA) detection immunized animal serum
The existence of antibody and concentration；After immunity completes, selecting to produce the sero-fast mouse of sufficiently high concentration, separating animal's spleen is also prepared
Splenocyte suspension；(see: K ǒ hler and Milstein, Nature, 215:495-497,1975 according to known hybridoma technology；
K ǒ hler et al, Immunology Today, 4:72-76,1983) obtained mouse boosting cell is blended with myeloma,
Prepare the sustainable hybridoma cell line passed on and secrete anti-PDPN monoclonal antibody.
The monoclonal antibody of two kinds of anti-PDPN, described monoclonal antibody belongs to IgG1 Subclass Antibodies, thin by above-mentioned hybridoma
Born of the same parents are generation, and the method preparing described monoclonal antibody has two kinds:
(1) in hybridoma culture fluids, above-mentioned hybridoma is inoculated, isolated and purified required specific anti-in nutrient solution after cultivation
The monoclonal antibody of PDPN；
(2) at the above-mentioned hybridoma of animal intraperitoneal inoculation, the specific anti-PDPN's separating and purifying required in animal ascites fluid
Monoclonal antibody.
Indirect enzyme-linked immunosorbent can be used to measure IgG concentration, and Western blot (Western-Blot) and streaming can be used further thin
Born of the same parents' art (FCM) detects the specific of gained monoclonal antibody.In the present invention, two strain of hybridoma the monoclonal produced resists
Body is named as SZ-163 and SZ-168 (seeing embodiment 3).
Through detecting by polyacrylamide electrophoresis method, the monoclonal antibody of the present invention belongs to IgG1 Subclass Antibodies.ELISA
Method testing result shows, the monoclonal antibody of the present invention can be with PDPN 22 peptide of Prof. Du Yucang, i.e. PLAG district and people liver
Cancer HepG2 cell-specific combines；Flow cytometry shows, the monoclonal antibody of the present invention can be special with multiple human cancer cell
Property combine, such as: NCI-H226 and transfected the Chinese hamster ovary celI of PDPN；It addition, our immunoblotting assay shows, this
Monoclonal antibody SZ163 and the SZ168 of invention are specific binding with the PDPN of reproducibility, with NCI-H226 cell pyrolysis liquid
It is combined in molecular weight 36kDa to go out in conjunction with band, is combined in molecular weight 67kDa with recombined human PDPN-Fc and goes out in conjunction with band (ginseng
See embodiment 3).
Our biological activity research shows, monoclonal antibody SZ-168 of the present invention can block expression PDPN effectively
Tumor cell induction hematoblastic activation assemble (seeing embodiment 4).
The functional monoclonal antibody SZ168 in the PLAG district of described anti-PDPN has the effect of platelet aggregation-against, is pressing down
Application in metastases processed.
The application in preparing medicine for anti transfer of tumor of the monoclonal antibody of described anti-PLAG.
The monoclonal antibody of described anti-PLAG is in the content setting up double antibody sandwich method mensuration human plasma solubility PDPN
Application.
The principle of the present invention is: the monoclonal antibody SZ168 in the PLAG district of anti-PDPN can be combined with the PLAG district of PDPN,
Thus having blocked the combination of blood platelet CLEC-2 and PLAG district, it is suppressed that it is little that PDPN with CLEC-2 is combined induced blood
Plate is assembled, and reaches to suppress the purpose of Nasopharyngeal neoplasms.
Experiment in vitro proves, the functional monoclonal antibody SZ168 of the anti-PDPN of the present invention, presses down with dosage-dependent manner
Human cancer cell processed and hematoblastic combination (seeing embodiment 4).
In vivo studies proves, the functional monoclonal antibody SZ168 of the anti-PDPN of the present invention can suppress tumour cell naked
Transfer (seeing embodiment 5) in mouse body.
Our double-antibody sandwich is tested monoclonal antibody SZ-163 as coated antibody, and SZ-168 marks horseradish peroxide
Compound enzyme is detection antibody (SZ-168-HRP), shows that monoclonal antibody SZ-163 of the present invention can be pressed from both sides with SZ-168-HRP
Heart success, detects recombined human PDPN, and with PDPN-Fc as standard items, draws out calibration curve (seeing embodiment 6).
Beneficial effect: the present invention compared with prior art has the advantage that
(1) monoclonal antibody of the present invention can be specific binding with people PDPN, and can effectively block people PDPN and hematoblastic knot
Close.
(2) the functional monoclonal antibody SZ-168 of the anti-PDPN of the present invention can suppress tumour cell life in nude mouse
Length and transfer.
(3) two kinds of monoclonal antibodies SZ-168 and the SZ-163 of the present invention can combine by epitopes different from PDPN, builds
Vertical double-antibodies sandwich ELISA, the plasma soluble PDPN level patients such as detection cancers provides a feasible side
Method.The level determination of plasma soluble PDPN can occur as tumour as a kind of new tumor markers and the diagnosis of transfer and pre-
Rear monitoring and differentiation.
Accompanying drawing explanation
The mass spectrogram of the PDPN22 peptide PDPN of Fig. 1 Prof. Du Yucang；
Fig. 2 ELISA method detection monoclonal antibody SZ-163 and SZ-168 are combined with Prof. Du Yucang PDPN polypeptide high specific；
It is specific binding with people PDPN that Fig. 3 Western-Blot blotting measures SZ-163 and SZ-168；A schemes: 18H5 is positive
Control antibodies, NCI-H226 is people's lung cancer in non-cellule type cell, and U87 is human glioma cell, and CHO-PDPN attaches most importance to
Group expresses the Chinese hamster ovary celI of people PDPN, and CHO is negative control cell.B schemes: SZ-163 and SZ-168 and recombined human PDPN-Fc
Fusion protein combines.
Fig. 4 flow cytometry detection monoclonal antibody SZ-168 is specific binding with people NCI-H226 and CHO-PDPN
Reaction；Mouse_IgG is negative control, and 18H5 is positive control.
Fig. 5 monoclonal antibody SZ-168 can dose-dependently suppress people lung cancer in non-cellule type cell NCI-H226 to induce
Platelet aggregation.
Fig. 6 monoclonal antibody SZ168 can suppress tumour cell transfer in nude mouse.Figure A shows monoclonal antibody
Subcutaneous and the growth of lung tumors and transfer in SZ168 suppression nude mouse, arrow indication is tumor growth position.Figure B is transfer
To subcutaneous tumor quality distribution map, comparing with control group, SZ-168 group significantly inhibits growth and the transfer of tumour, P < 0.001.
Fig. 7 SZ163 detects the calibration curve of solubility PDPN, PDPN-Fc with SZ168 double antibody sandwich ELISA
For standard items.
Detailed description of the invention
Below in conjunction with the accompanying drawings and embodiment the invention will be further described:
Embodiment 1: immunogene prepares the 22 amino acid whose polypeptide fragments in the PLAG district of Prof. Du Yucang people PDPN
(DTETTGLEGGVAMPGAEDDVVC) (mass spectrogram is shown in Fig. 1) becomes with keyhole limpet hemocyanin (KLH) coupling
DTETTGLEGGVAMPGAEDDVVC-KLH is as immunogene, immunity Balb/c mouse.(entrust purple territory, Shanghai biological
Science and Technology Ltd. completes)
Embodiment 2: the preparation of the monoclonal antibody in the PLAG district of specific anti-PDPN
We apply conventional immunological method and hybridoma technology (K ǒ hler and Milstein, Nature, 215:495-497,1975)
The monoclonal antibody of the preparation present invention.
First, with coupling 22 peptide-KLH (DTETTGLEGGVAMPGAEDDVVC-KLH) immunity of Prof. Du Yucang
8 week old female Balb/c mouse (Shanghai academy of sciences Experimental Animal Center), altogether immunity three times, every minor tick surrounding.First twice is the back of the body
The subcutaneous multi-point injection in portion adds lumbar injection, and third time is for add lumbar injection through mouse tail vein injection.
Until reached by the serum antibody titer of immune mouse sufficiently high after, put to death animal separating spleen cell.Application standard
Monoclonal Antibody Cell integration technology (K ǒ hler and Milstein, Nature, 215:495-497,1975), will be by immunity Balb/c
The spleen cell of mouse carries out cell with SP2/0 myeloma cell's (introduction of Paris, FRA blood Collection Center hybridoma laboratory) of mouse
Merge.In HAT culture medium select cultivate fused cell, take after cultivation supernatant with ELISA method filter out high-level secretory resist
The cell line of body.Cultivate or frozen for further expanding.
Application ELISA method, FCM and western blot method etc. are biochemical screens and after qualification with immunological technique, it is thus achieved that two strains
The monoclonal antibody in the PLAG district of specific anti-PDPN, is respectively designated as SZ-163 and SZ-168.
In order to prepare monoclonal antibody in a large number, we select BALB/c mouse, first inject with norphytane row mouse peritoneal, one
After week, hybridoma is inoculated in mouse peritoneal (5 × 105/ mouse).About obvious ascites is i.e. had to produce after inoculating one week,
Every mouse can collect 5～10ml ascites.
Embodiment 3: the biochemical property of monoclonal antibody of the present invention
(1) application polyacrylamide electrophoresis method measures and confirms, monoclonal antibody SZ-163 and SZ-168 of the present invention belongs to IgG1 subclass.
(2) it is inoculated in Balb/c mouse peritoneal generation ascites by producing the monoclonal antibody SZ-163 clone strain with SZ-168, uses
Protein G-Sepharose-4B (Pharmacia companyProduct) post purifying, every 1mL ascites can purify and obtain IgG 1.0mg.
(3) ELISA measuring SZ-163 with SZ-168 is specific: by 22 peptides (1 μ g/ml) of the PDPN of Prof. Du Yucang
4 DEG C are coated overnight, after PBS-0.05%-polysorbas20 washs, close overnight with 2%BSA-PBS.By SZ-163 and SZ-168
From 6 concentration of concentration doubling dilution of 1 μ g/ml (1 μ g/ml, 500ng/ml, 250ng/ml, 125ng/ml, 62.5ng/ml,
31.25ng/ml), 37 DEG C are reacted 1.5 hours.After washing, add the sheep anti-mouse antibody of horseradish peroxidase-labeled
(Beckman-Immunotech Products), after 37 DEG C are reacted 1 hour, then adds TMB colour developing.Enzyme-linked immunosorbent assay
Measuring display, SZ-163 and SZ-168 is combined with Prof. Du Yucang PDPN polypeptide high specific.(Fig. 2)
(4) immunoblot assay (Western-blot) determines the antigen molecule of monoclonal antibody identification of the present invention: recombined human PDPN-Fc,
U87 cell pyrolysis liquid, the Chinese hamster ovary celI lysate expressing restructuring PDPN and NCI-H226 cell pyrolysis liquid are through 10%
SDS-PAGE separates, and electrotransfer is on pvdf membrane.Room temperature closes 1h, pvdf membrane and monoclonal antibody with the skimmed milk power of 5%
(3 μ g/ml) 4 DEG C overnight, then makes the sheep anti-mouse antibody room temperature reaction 1 hour of the monoclonal antibody of combination and horseradish peroxidase-labeled,
Then substrate colour developing is carried out with Enhanced chemiluminescence (ECL).Western-blot Blot experiment confirms, SZ-163 and SZ-168
Can be combined with the PDPN of reproducibility, be combined in molecular weight 36kDa with people's lung cancer in non-cellule type NCI-H226 cell pyrolysis liquid
Go out in conjunction with band, be combined in molecular weight 67kDa with recombined human PDPN-Fc and go out in conjunction with band, with human glioma cell U87
It is combined in molecular weight 46kDa with CHO-PDPN cell pyrolysis liquid to go out in conjunction with band (Fig. 3).This result proves SZ-168 further
Be combined with people's PDPN high specific with SZ-163.
[5] flow cytometry (FCM) determines that SZ-168 can be specific binding with the kinds of tumor cells expressing PDPN:
The Chinese hamster ovary celI cultivating NCI-H226 cell and express PDPN, collects cell with 0.25% pancreas enzyme-EDTA digestion, and cell is dense
Degree is 1*106/ pipe, antibody with the amount of 2 μ g/ pipes and cell incubation, room temperature 1 hour, washs 1 time with PBS, adds PE-
The fluorescence two of sheep anti-Mouse resists, incubated at room 1 hour, upper machine testing after washing 2 times with PBS.FCM result shows, SZ-168
(Fig. 4) can be reacted with above-mentioned cell-specific.
Embodiment 4: monoclonal antibodies block platelet aggregation induced by tumor cells of the present invention
Gather normal healthy people whole blood (3.8% sodium citrate 1/9 anti-freezing) 100 × g to be centrifuged 10 minutes, take supernatant platelet-rich blood plasma
(PRP).The monoclonal antibody (5-10 μ g/ml) of various concentration is hatched 15 points the most on ice with human tumor cells NCI-H226 respectively
Clock, the most respectively with human tumor cells NCI-H226 cell (1*107/ ml) induced platelet aggregation.Platelet aggregation test
Result shows, anti-PDPN monoclonal antibody SZ-168 can suppress the human platelet aggregation (Fig. 5) of tumor cell induction.
Embodiment 5 monoclonal antibody SZ-168 can suppress tumour cell transfer in nude mouse
The nude mice (Shanghai academy of sciences Experimental Animal Center) in 5-8 week is divided into experimental group and control group, CHO-PDPN cell (1*106/
Nude mice) internal through tail vein injection to two group, control group injection is to hatch the CHO-PDPN of 30 minutes on ice with PBS
Cell, experimental group injection is the CHO-PDPN cell hatched on ice with SZ-168 (100ug/100ul), from injection day
Start to observe mouse state, when nude mice occurs that action edge is slow, during slow respiration etc., put to death mouse, count nude mice by subcutaneous, lung
The number of portion's tumor nodule, quality and volume, and do statistical analysis (Fig. 6, table 1).Result shows monoclonal antibody SZ168
Subcutaneous and the growth of lung tumors and transfer in suppression nude mouse.
Table 1:
Embodiment 6: monoclonal antibody SZ-163 and SZ-168 set up double antibody sandwich method and measure the content of human plasma solubility PDPN
By monoclonal SZ-163 (coated antibody) with 10 μ g/ml wrapper sheets on 96 hole ELISA Plates, 100ul/ hole, 4 DEG C are overnight, warp
After the washing of PBS-0.05%-polysorbas20, the BSA-PBS with 2% closes overnight, with recombined human PDPN-Fc as standard items, with
The concentration doubling dilution of 125ng/ml prepares calibration curve 7 point, and every hole adds 100ul human plasma sample or standard items, 37 DEG C of reactions
1 hour；In conjunction with PDPN and horseradish peroxidase-labeled monoclonal antibody SZ-168 (sodium periodate oxidizing process, SZ168-HRP)
37 DEG C are reacted 1 hour.Develop the color for substrate with TMB, detect absorbance (Fig. 7) at 450 nm.Result shows monoclonal
Antibody SZ-163 Yu SZ-168 sets up double antibody sandwich method (ELISA) can measure the content of human plasma solubility PDPN,
Can occur as tumour as a kind of new tumor markers and the diagnosis of transfer and prognostic monitoring and differentiation.
<110>
First Affiliated Hospital of Soochow University,Suzhou
<120>
The monoclonal antibody of anti-human flatfoot proteinemina platelet accumulation regions and application thereof
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1
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PatentIn version 3.3
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1
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PRT
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Artificial sequence
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1
Asp Thr Glu Thr Thr Gly Leu Glu Gly Gly Val
Ala Met Pro Gly Ala
1 5 10 15
Glu Asp Asp Val Val Cys
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Claims (9)
- The hybridoma cell strain of the most anti-PLAG, its deposit number is respectively CCTCC NO:C201613 and CCTCC NO:C2015185.
- The monoclonal antibody of the most anti-PLAG, respectively by secreted by the hybridoma cell line that deposit number is CCTCC NO:C201613 and CCTCC NO:C2015185, named SZ-163 and SZ-168 of described monoclonal antibody.
- The monoclonal antibody of anti-PLAG the most according to claim 2, it is IgG1 Subclass Antibodies.
- The monoclonal antibody of anti-PLAG the most according to claim 2, it can be specific binding with multiple human cancer cell.
- The monoclonal antibody of anti-PLAG the most according to claim 4, it is specific binding with the PDPN of reproducibility, is combined in molecular weight 36kDa with NCI-H226 cell pyrolysis liquid and goes out in conjunction with band, is combined in molecular weight 67kDa with recombined human PDPN-Fc and goes out in conjunction with band.
- 6. the preparation method of the monoclonal antibody of anti-PLAG described in claim 2, it is characterized in that, inoculating deposit number in hybridoma culture fluids is CCTCC NO:C201613 and the hybridoma of CCTCC NO:C2015185, the isolated and purified monoclonal antibody obtaining required anti-PLAG in nutrient solution after cultivation；OrIt is CCTCC NO:C201613 and the hybridoma of CCTCC NO:C2015185 at animal intraperitoneal inoculation deposit number, the monoclonal antibody of the anti-PLAG separating and purifying required in animal ascites fluid.
- 7. the monoclonal antibody of anti-PLAG application in preparing medicine for anti transfer of tumor described in claim 2.
- 8. the application in the content setting up double antibody sandwich method mensuration human plasma solubility PDPN of the monoclonal antibody of anti-PLAG described in claim 2.
- Application the most according to claim 8, it is characterised in that using monoclonal antibody SZ-163 as coated antibody, and SZ-168 mark horseradish peroxidase is detection antibody, detects recombined human PDPN, and with PDPN-Fc as standard items, draws out calibration curve.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106947744A (en) * | 2017-03-14 | 2017-07-14 | 福州迈新生物技术开发有限公司 | The hybridoma cell strain of one plant of anti-flatfoot protein monoclonal antibody of secretion and its application |
| WO2019081714A1 (en) | 2017-10-26 | 2019-05-02 | Vib Vzw | Podoplanin positive macrophages |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106947744A (en) * | 2017-03-14 | 2017-07-14 | 福州迈新生物技术开发有限公司 | The hybridoma cell strain of one plant of anti-flatfoot protein monoclonal antibody of secretion and its application |
| CN106947744B (en) * | 2017-03-14 | 2020-05-05 | 福州迈新生物技术开发有限公司 | Hybridoma cell strain secreting monoclonal antibody against podophyllum protein and application thereof |
| WO2019081714A1 (en) | 2017-10-26 | 2019-05-02 | Vib Vzw | Podoplanin positive macrophages |
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| CN105754953B (en) | 2019-06-25 |
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