CN102219854A - Monoclonal antibody of anti-human PIGF (placental growth factor) protein as well as preparation method and application thereof - Google Patents
Monoclonal antibody of anti-human PIGF (placental growth factor) protein as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody of anti-human PIGF (placental growth factor) protein, which can be specifically bonded with human PIGF and block PIGF from joining receptors of the PIGF. The invention further discloses a preparation method of the monoclonal antibody. Besides, the invention discloses an application of the monoclonal antibody in qualitative and quantitative detection to the human PIGF protein. Furthermore, the invention discloses an application of the monoclonal antibody in preparing drugs for treating tumors.
Description
Technical field
The invention belongs to biotechnology-monoclonal antibody field.The present invention relates to the proteic monoclonal antibody of a kind of anti-people PlGF; In addition, the invention still further relates to this MONOCLONAL ANTIBODIES SPECIFIC FOR method and uses thereof.
Background technology
Angiogenesis or hyperplasia (angiogenesis) are being meant that biologically intravital already present blood vessel (as capillary vessel and Venule) is by sprouting or the splitted mode produces the process of new blood vessel.Angiogenesis is useful and essential in the many normal physiological process of keeping body as organizing that fetal development, trauma wounds De More close and repair etc.But over-drastic angiogenesis or hyperplasia are also closely bound up with many pathological changes (as the hyperplasia diffusion of tumour, inflammation).
In the body blood vessel can be newborn or outgrowth key be that the vascular endothelial cell that is its liner has division and proliferation and directional migration is inserted the ability of existing vascular wall.In the factor of all kinds of stimulation vascular endothelial proliferations, with vascular endothelial growth factor (vascular endothelialgrowth factor, VEGF) particularly important.In fact, VEGF is the factor of at present known strong impulse vascular endothelial cell division and proliferation.The importance of VEGF in stimulating blood vessel hyperplasia also is proved in the research of VEGF gene knockout mice: as needing only after the portion in the VEGF gene is knocked out, blood vessel among the embryo promptly can't normally form and then cause embryo's death when growing to 11 to 12 days (Nature 1996 such as Carmeliet P, 380:435; Nature 1996 such as Ferrara N, 380:439).
VEGF is general often synthesize by vascular endothelial cell, scavenger cell and tumour cell, and by autocrine/paracrine mode acceptor on the vasoactive endotheliocyte specifically, and bring into play effects such as promotion vascular endothelial cell growth, propagation, migration, formation.Studies show that, in the cancerous tissue of most of malignant tumour patient, also can detect VEGF, and the expression level of VEGF increases relevant (Dvorak HF etc.: J Exp Med 1991 with malignancy of tumor degree and progression of disease; 174:1275-8; Cancer Res 1993 such as BrownLF; 53:4727-35; Weidner N, Semple JP, Welch WR and Folkman J:N Engl J Med 1991; 324:1-8.4-5); In addition, VEGF plays an important role in the blood vessel of inflammation pathology, increases as VEGF level in (rheumatoid synovial fluids and tissue) and ox-hide moss (psoriasis) tissue of patient in sacroiliitis patient's knuckle synovia and tissue.VEGF in the blocking-up body, thus stop by VEGF and receptor-mediated short blood vessel hyperplasia thereof, can reach and suppress tumor growth and transfer, or the effect of anti-inflammatory.
Suppressing VEGF and receptor-mediated blood vessel hyperplasia medicament research and development field thereof, the main at present two big means that exist: promptly
1) seek the material can directly suppress intrinsic protein tyrosine kinase activity in the vegf receptor, if the drug main of such research and development various small molecules thing inhibitor such as PTK787/ZK 222584 (Cancer Res 2000 such as WoodJM, 60:2178);
2) by suppressing combining of VEGF and its acceptor, stop VEGF and acceptor thereof in the effect of the short vascular endothelial cell division and proliferation of mediation and the effect of inhibition blood vessel hyperplasia thereby reach, the medicine of such research and development comprises the antibody of various anti-VEGF or anti-vegf receptor, (Nature 1993 such as Kim KJ, 362:841 such as antisense oligonucleotide and micromolecular inhibitor; Cancer Res such as Presta LG, 1997,57:4593; Clinical Cancer Research such as Posey J A, 2003,9:1323).The Bevacizumab (trade(brand)name Avastin) that is is wherein researched and developed, produced and obtained in 2004 the drugs approved by FDA list marketing by U.S. Genentech company is exactly a kind of identification and the humanized monoclonal antibody that combines VEGF.Avastin is by neutralization or remove that VEGF reaches the inhibition tumor vascular growth in the body, and then the effect of containment growth of tumor and transfer (Cancer Res such as Presta LG, 1997,57:4593; N Engl J Med such as Hurwitz H, 2004; 350:2335).
The predecessor of humanized antibody Avastin can trace back to the mouse monoclonal antibody that code name is A4.6.1.The source of this A4.6.1 mouse monoclonal antibody and the hybridoma cell line and the purposes of secreting it are 6 in the patent No., 582,959 United States Patent (USP) (contriver: Kim, Kyung Jin, patent disclosure date: June 24,2003, patent name: Antibodies to vascular endothelialcell growth factor); And the patent No. is that (contriver: Kim, the Kyung Jin patent disclosure date: June 5,2007, patent name: all describe to some extent Antibodies tovascular endothelial cell growth factor) for 7,227,004 United States Patent (USP).But also there is following deficiency in this antibody: (1) is the same with most monoclonal antibody, A4.6.1 mouse monoclonal antibody and humanized antibody Avastin thereof also just can be in conjunction with the antigenic part of VEGF sites, can't be familiar with or contain to cover VEGF and go up numerous his antigen sites.(2) further experimentation on animals and clinical study are found only to inject A4.6.1 mouse monoclonal antibody or its humanized antibody Avastin separately, can't reach the angiogenesis promoting that neutralization fully suppresses interior VEGF of body and mediation thereof.
A recent experimentation on animals report is treated insensitive tumour (being the VEGF-resistant tumour) to some anti-VEGF antibodies, by injecting mouse anti mouse placenta growth factor (placental growth factor, PlGF) antibody, (see Fischer C etc.: Cell 2007,131:463-475) also can to reach the effect that suppresses the tumor vessel hyperplasia and suppress tumor growth and transfer.
PlGF and VEGF homology, structure and biological activity are close, and all can with the VEGFR1 receptors bind.But that research is in the past more paid close attention to is VEGF, and for PlGF, (Maglione D et al.PNAS 1991,88:9267-9271), its meaning in blood vessel hyperplasia is to be in subordinate or unsettled condition for a long time always although its encoding gene just cloned as far back as 1991.Along with going deep into of studying, increasing result proves that in fact PlGF participates in the blood vessel hyperplasia under many physiology or the pathological conditions in recent years.Support the evidence of this conclusion comprise at least following some:
1) PlGF is except high level expression in placenta, also detects the PlGF expression of gene in other histoorgans in the body (as lungs etc.) and the cell;
2) its angiogenesis that after wound or transplantation tumor, is brought out of the mouse of PlGF gene knockout than its in normal mouse, obviously reduce (Carmeliet P et al, Nature Medicine2001,7:575-583);
3) the insane generation of the tendency among PlGF and solubility FLT-1 acceptor (being VEGFR-1) proteic level and high risk pregnancy women son is closely related with development in blood or the urine;
4) as previously mentioned, experimentation on animals proved the injection mouse-anti-mouse PlGF antibody after, tumor vessel hyperplasia in the mouse and tumor growth can obtain certain control (Fischer C etc.: Cell2007,131:463-475).
And on this basis, as further develop a kind of can in conjunction with and neutralization suppress the proteic monoclonal antibody of people PlGF, then this monoclonal antibody can be used as the separate constituent that blood vessel hyperplasia suppresses medicine, or be used in combination with antibody Avastin that other blood vessel hyperplasias suppress medicines such as anti-people VEGF, reach the purpose of the short blood vessel hyperplasia that suppresses PlGF and VEGF mediation more completely.Such monoclonal antibody is in the fundamental research of blood vessel hyperplasia and all will have remarkable meaning and widespread use value clinically.
Summary of the invention
One of the technical problem to be solved in the present invention provides the proteic monoclonal antibody of a kind of anti-people PlGF, but this monoclonal antibody specific combination people PlGF albumen, and the combining of blocking-up PlGF and its acceptor VEGFR1.
Two of the technical problem to be solved in the present invention provides the preparation method of said monoclonal antibody.
Three of the technical problem to be solved in the present invention provides said monoclonal antibody proteic application of people PlGF in the detection by quantitative biological sample.
Four of the technical problem to be solved in the present invention provides said monoclonal antibody proteic application of people PlGF in the qualitative detection biological sample.
Five of the technical problem to be solved in the present invention provides the application of said monoclonal antibody in the medicine of preparation treatment tumour.
It is immunogen that the present invention chooses recombinant human PlGF albumen, by the subcutaneous immunity of the mouse of repeated multiple times low dose, obtains the high anti-people PlGF protein polyclone antibody of tiring of secretion; Picking mouse therefrom again, get its spleen cell,, be respectively P16 comprising a plurality of code names by the external hybridoma monoclonal cell that merges, set up through steps such as drug screening and subclones the more anti-people PlGF of stably excreting antibody with murine myeloma cell, P5, P20, P28, P38, P61, the hybridoma cell strain of PV19 and P21-2 is identified through several different methods such as ELISA, immunohistochemical methodss, confirms that secreted separately monoclonal antibody can specific combination people PlGF albumen.The P16 monoclonal antibody that also proves the external biological activity research can suppress combining of PlGF and its acceptor (being VEGFR1).
In one aspect of the invention, provide the proteic monoclonal antibody of a kind of anti-people PlGF (code name is P16), but this mono-clonal specific combination people placenta growth factor PlGF, and the combining of blocking-up PlGF and its acceptor VEGFR1.
In another aspect of this invention, provide a kind of preparation method of said monoclonal antibody, comprise the steps:
Step 5, positive hybridoma cell are identified the hybridoma that obtains the proteic monoclonal antibody of the anti-people PlGF of stably excreting through subclone;
Step 6, hybrid tumor cell amplification is cultivated, collected nutrient solution, adopt the proteic monoclonal antibody of the anti-people PlGF of affinity chromatography separation and purification.
Wherein, step 1 is specially: respectively with pcr amplification obtain the encoding gene segment of people PlGF131, it is cloned in the Yeast expression carrier pPic9K carrier, obtain expression plasmid pPic9K-PlGF131, transform Pichia yeast, filter out efficient expression engineering strain, engineering strain is after fermentation, abduction delivering, separation and purification, and acquisition purity reaches the recombinant human PlGF131 albumen more than 95%.
Wherein, the method for the subcutaneous immunity of the described mouse of step 2 is: (Freund's complete adjuvant is a kind of water in oil emulsion, can very effectively induce to produce high titer antibody with the recombinant human PlGF albumen of described purifying and Freund's complete adjuvant; Freund's complete adjuvant contains the cell wall constituent of mycobacterium tuberculosis, can strengthen antigenic antibody response; Adjuvanticity comes from immunogenic lasting release in the oil droplet, and stimulates the local immunity reaction) mix, in subcutaneous multi-point injection mouse.
Wherein, the described affinity chromatography of step 6 is specially: the hybridoma filtrate supernatant that will contain monoclonal antibody is splined in the proteic 4% agarose particulate pearl of reorganization protein-A (the protein-A agarose beads) affinity chromatographic column that has been pre-charged with covalent cross-linking; After last sample finished, affinity chromatographic column was removed foreign protein with the PBS wash-out, again the antibody protein that is adsorbed by the particulate pearl with glycine liquid wash-out, dialysis again; Dialyzed sample promptly obtains the proteic monoclonal antibody of anti-people PlGF of purifying more after filtering.
In another aspect of this invention, provide a kind of said monoclonal antibody in the application that quantitatively reaches in the qualitative detection people PlGF albumen.(as vitamin H, radiation element or marks such as fluorescein such as FITC) or unlabelled this monoclonal antibody of mark are the probe photographic developer, be used for the PlGF expression level that immunohistochemical methods, immunofluorescence, radioautograph or additive method come the histoorgan of qualitative location or detection by quantitative various diseases such as tumour or inflammatory patients, with auxiliary clinical diagnosis.
In another aspect of this invention, provide the application of a kind of said monoclonal antibody in the medicine of preparation treatment tumour.Monoclonal antibody of the present invention can be used as the proteic antagonist pharmaceuticals composition of people PlGF, is used for the treatment of the multiple disease that causes because of the blood vessel hyperplasia and comprises various malignant tumours, human breast carcinoma for example, colorectal carcinoma and rhabdomyoma etc.
In addition, secrete the hybridoma cell line of this antibody, can be used to separation and purification mRNA, therefrom clone and amplify the light chain of encoding antibody and the gene of variable region of heavy chain again with methods such as RT-PCR.Light chain of antibody and heavy chain variable region gene that amplification obtains can be used for preparing range gene engineered antibodies such as single-chain antibody, Fab segment, people-mouse chimeric antibody or humanized antibody.This antibody-like or its redundant organism (as antibody fragment, chimeric antibody, humanized antibody, radiopharmaceuticals traget antibody etc.) are as the drug alone composition, or merge to use with other drug, can be used for preparing the medicine or the preparation of the pathology relevant (as the hyperplasia diffusion of tumour, inflammation etc.) with the PlGF high expression level.
Description of drawings
The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments:
Fig. 1 is with the experimental result synoptic diagram of enzyme linked immunosorbent assay (ELISA) in the external evaluation recombinant human of 96 orifice plates PlGF131 albumen (hPlGF131) and VEGFR1 receptors bind among the embodiment 1, the wherein positive contrast of recombinant human VEGF 165 albumen (hVEGF165), bovine serum albumin (BSA) is as negative control.
Fig. 2 A and Fig. 2 B are with monoclonal antibody and the protein bound experimental result synoptic diagram of recombinant human PlGF that is coated on 96 orifice plates in the culture supernatant of ELISA method detection hybridoma among the embodiment 2.Wherein, hybridoma cell strain P5 among Fig. 2 A, P16, all contain in the culture supernatant of PV19 and P20 can with the protein bound monoclonal antibody of recombinant human PlGF; Do not contain protein bound monoclonal antibody in the culture supernatant of hybridoma cell strain M23 with recombinant human PlGF; The murine myeloma cell culture supernatant that the SP2/0 representative is not merged, negative contrast.Hybridoma cell strain P5 among Fig. 2 B, P21-2, P28, all contain in the culture supernatant of P38 and P61 can with the protein bound monoclonal antibody of recombinant human PlGF.
Fig. 3 is the protein bound experimental result synoptic diagram of recombinant human PlGF that detects monoclonal antibody P16 and DTT sex change and non-DTT sex change among the embodiment 3 with immuning hybridization (western blot) method.Wherein band 1 and 2 sample are the people PlGF albumen of purchasing in U.S. R and D Systemes company, and band 3 and 4 sample are for press the people PlGF albumen that separates acquisition among the embodiment 1.
Fig. 4 combines the proteic experimental result synoptic diagram of the recombinant human PlGF that is coated on 96 orifice plates for the P16 monoclonal antibody that detects different solubility with the competitive ELISA method among the embodiment 4 external the competition with fixing biotin labeled soluble VEGFR 1 acceptor of solubility-Fc fusion rotein (Biotin-VEGFR1-Fc).
Fig. 5 is the experimental result synoptic diagram that the proteic monoclonal antibody P16 of anti-people PlGF suppresses the growth of people A673 rhabdomyoma among the embodiment 5 in nude mouse.
Fig. 6 is with the different proteic monoclonal antibody (P5 of anti-people PlGF among the embodiment 6, P21-2, P61, P28 and P38) be coated antibody, with biotin labeled P16 monoclonal antibody serves as to detect antibody, with the experimental result synoptic diagram of the outer detection by quantitative people PlGF albumen of sandwich ELISA body of laws with reference to product.
Fig. 7 serves as to detect antibody with biotin labeled P61 monoclonal antibody, with the experimental result synoptic diagram of the outer detection by quantitative people PlGF albumen of sandwich ELISA body of laws with reference to product for being coated antibody with the proteic monoclonal antibody P16 of anti-people PlGF among the embodiment 7.
Fig. 8 is for being detection reagent with the P16 monoclonal antibody among the embodiment 8, detects the Chinese hamster ovary celI (A) of people PlGF gene transfection, people's healthy tissues section (B: colon with the immunohistochemical methods method; The C: (D: human breast carcinoma tissue of stomach-tissue and histopathologic slide; E: human colon carcinoma tissue; F: the human colon carcinoma tissue, under high power) in the result schematic diagram of microscopically observation.
Embodiment
The following examples can make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.
Embodiment 1: proteic expression preparation of the preparation of immunizing antigen: recombinant human PlGF131 and external activity are identified
It is 200810018884.8 that application number is adopted in the proteic expression preparation of recombinant human PlGF131, the method preparation of denomination of invention for describing in the Chinese invention patent application of " expression production, separation and purification and the chemical labeling thereof of recombinant placenta growth factor ".
Utilize round pcr from human lung tissue's cell cdna library, to increase respectively and obtain encoding complete open reading frame sequence (the open-reading frame of people PlGF131, ORF) gene segment, identify correct through sequencing, after the restriction enzyme processing, it is cloned in Yeast expression carrier pPic9K (Invitrogen company) carrier.Obtain expression plasmid pPic9K-PlGF131.Transform Pichia yeast through electricity again, filter out efficient expression engineering strain.Engineering bacteria, obtains purity respectively and reaches recombinant human PlGF131 albumen more than 95% after the separation and purification through fermentation, abduction delivering.
The proteic external activity of step 2. recombinant human PlGF131 is identified
All can combine in view of people PlGF albumen is the same with people's vegf protein, adopt in the present embodiment, earlier in the people PlGF131 albumen of the above-mentioned acquisition of external evaluation and the activity of VEGFR1 receptors bind at 96 orifice plates with the VEGFR1 receptor-specific.(it is 200710047352.2 that application number is adopted in the proteic expression preparation of this VEGF165 with the positive contrast of people VEGF165 albumen respectively, the method preparation of denomination of invention for describing in the Chinese invention patent application of " separation and purification of recombinant human vascular endothelial growth factor, chemical labeling in vitro and application thereof "), (every hole bag is 5ug/ml by solubility by 96 orifice plates for people PlGF131 albumen and negative control protein B SA (bovine serum albumin) bag, pH 9.6,0.1M NaHCO3 liquid), 4 ℃ are spent the night; The sealing of 2% bovine serum albumin, the soluble human vegf R1 receptor protein (available from U.S. R and DSystmes company) of the different solubility of adding was hatched 1 hour for 37 ℃ after the washing of PBS-0.1%Tween20 liquid.After the washing of PBS-0.1%Tween20 liquid, add mouse-anti human VEGFR-3 1 receptor antibody (available from U.S. R and D Systmes company), hatched 1 hour for 37 ℃.After the washing of PBS-0.1%Tween20 liquid, add the goat anti-mouse ig antibody (U.S. Sigma company product) of horseradish peroxidase (HRP) mark, hatched 1 hour for 37 ℃.Behind PBS-0.1%Tween20 liquid thorough washing, add O-Phenylene Diamine (OPD)-0.1%H again
2O
2Substrate solution colour developing 10-15min is with 0.1M HCl termination reaction.In MK3-Multiskan microplate reader (Thermo Scientific company product), read the OD of 492nm place value (OD at 492nM).The result is as shown in Figure 1: the same with recombinant human VEGF 165 albumen (hVEGF165), recombinant human PlGF131 albumen (hPlGF131) also keeps the activity with the VEGFR1 receptors bind.
Embodiment 2: the foundation and the Screening and Identification of the hybridoma cell line of the proteic monoclonal antibody of the anti-people PlGF of stably excreting
The recombinant human PlGF131 albumen of the foregoing description 1 purifying is mixed with Freund's complete adjuvant (available from U.S. Sigma company), in subcutaneous multi-point injection C57BL/6 mouse (Shanghai Slac Experimental Animal Co., Ltd., 100ul/ only is total to 10ug PlGF131 albumen).First immunisation 2-3 is after week, and mouse gives subcutaneous multi-point injection people PlGF131 albumen and Freund (available from U.S. Sigma company) mixed solution booster immunization again.Behind booster immunization 2-3 time, the mice serum that takes a morsel, with bag by the proteic 96-of people PlGF131 hole enzyme plate detect tiring of anti-PlGF131 protein antibodies in the mice serum with the ELISA method, the high person's mouse boosting cell of tiring is used for next step cytogamy.
Back 3 days of people PlGF131 albumen last immunity, anesthesia and the aseptic mouse spleen that takes out down, spleen grinds and hypotonic processing removal red corpuscle through glass, after under the 1000rpm rotating speed centrifugal 3 minutes, mouse boosting cell heavily is dissolved in physiological saline again, obtains the mouse boosting cell suspension.The mouse boosting cell suspension that obtains merges under 50%PEG-1500 (U.S. Sigma company product) effect with 10: 1 ratios with SP2/0 murine myeloma cell (available from school of life and health sciences cell preservation center, Chinese Academy of Sciences Shanghai) again.Merge method (Kohler G.and Milstein C:Nature1975 routinely; 256:495-497) carry out, PEG consumption 1ml slowly added in 1 minute.React after 90 seconds, RPMI-1640 substratum (available from American I nvitrogen company) termination reaction with serum-free, the centrifugal 10min of 1000rpm, remove supernatant liquor, again the cell under the centrifugation is adjusted to 1X10 with the RPMI-1640-10%FCS substratum (FCS is available from American I nvitrogen company) that contains 10%HAT (H is the amino dish purine of xanthoglobulin, A, T thymidine, is Sigma company product) with cell concn
6/ ml adds the flat Tissue Culture Plate in 96 holes (every hole 200ul), in 37 ℃, cultivates 2-3 week in the 5%CO2 incubator.
With recombinant human PlGF131 albumen (5ug/ml, pH 9.6,0.1M NaHCO3 liquid) coated elisa plate, 4 ℃ are spent the night; 2% bovine serum albumin (BSA) sealing.After the washing of PBS-0.1%Tween20 liquid, add Hybridoma Cell Culture supernatant to be checked (with the negative contrast of SP2/0 murine myeloma cell culture supernatant of not merging) and hatched 2 hours for 37 ℃.After the washing of PBS-0.1%Tween20 liquid, add the goat-anti mouse Ig (Sigma company product) of horseradish peroxidase (HRP) mark, hatched 1 hour for 37 ℃.Behind PBS-0.1%Tween20 liquid thorough washing, add O-Phenylene Diamine (OPD)-0.1%H again
2O
2Substrate solution colour developing 10-15min is with 0.1M HCl termination reaction.In MK3-Multiskan microplate reader (U.S. Thermo Scientific company product), read the OD of 492nm place value.The OD value that records is than the high 5-10 of negative control hybridoma cloning more doubly, and it is frozen to increase.
Cloning-the limiting dilution assay of step 4, positive hybridoma cell
The positive cell that above-mentioned primary dcreening operation is obtained is diluted to every hole 1-10 cell with the RPMI-1640-10%FCS substratum, is laid on 96-porocyte culture plate, in 37 ℃, cultivates 2-3 week in the 5%CO2 incubator.Treat that the clone grows up to, get supernatant liquor and detect the secretion of identifying anti-people PlGF protein antibodies with ELISA once more.
Fig. 2 A and Fig. 2 B detect with the ELISA method to identify hybridoma supernatant and the proteic synoptic diagram that combines of recombinant human PlGF131.Fig. 2 A result shows P5, P16, all contain in the hybridoma cell strain supernatant liquors such as P20 and PV19 can with the protein bound monoclonal antibody of recombinant human PlGF131; Do not contain protein bound monoclonal antibody in the culture supernatant of hybridoma cell strain M23 and the myeloma cell SP2/0 that do not merge with recombinant human PlGF.Fig. 2 B result shows hybridoma cell strain P5 and hybridoma cell strain P21-2, P28, all contain in the culture supernatant of P38 and P61 can with the protein bound monoclonal antibody of recombinant human PlGF.
Synthesizing map 2A and Fig. 2 B detected result prove that obtaining many strains secretes the hybridoma cell strain of the proteic monoclonal antibody of anti-people PlGF.
More than each hybridoma cell strain again after subcloning and amplification in vitro are cultivated, go down to posterity for a long time and frozen preservation.
The in-vitro separation purifying and the detection of the proteic monoclonal antibody of embodiment 3. anti-people PlGF
Step 1:, treat that cell concn reaches 10 with hybridoma amplification cultivation in serum free medium that the foregoing description 2 is set up
5Stop liquid feeding when/ml is above, continue again to cultivate after the cell culture fluid flavescence, to collect nutrient solution, centrifugal 10 minutes of 1500rpm, supernatant liquor preserve standby or are directly used in the separation and purification of next step monoclonal antibody in 4 ℃ or-20 ℃ behind 0.45 μ m membrane filtration again.
Step 2: the protein-A affinity chromatography is adopted in the separation and purification of the proteic monoclonal antibody of anti-people PlGF.Its purification step is: the above-mentioned hybridoma filtrate supernatant that contains the proteic monoclonal antibody of anti-people PlGF is splined on proteic 4% agarose particulate pearl (the protein-A agarose beads of reorganization protein-A that has been pre-charged with covalent cross-linking, available from GE, i.e. GE company) in the affinity chromatographic column; After last sample finished, affinity chromatographic column was removed foreign protein with the PBS wash-out, again the antibody protein that is adsorbed with low pH (2.7) glycine (0.1M) liquid wash-out.Elutriant is regulated pH to 7.0 with 1mol/L Tris (pH 9.0), to the 1xPBS dialysis of 10 times volume after 12～16 hours (during change liquid 2-3 time), dialyzed sample promptly obtains the proteic monoclonal antibody of anti-people PlGF of purifying again behind 0.45 μ m membrane filtration again.
SDS-PAGE (vitriol-polyacrylamide gel electrophoresis of sodium dodecyl) analytical proof, gained monoclonal antibody product purity is greater than 95%.These samples are used for following further analysis and research.
In the present embodiment, detect the monoclonal antibody P16 of purifying and the proteic specific combination of recombinant human PlGF of DTT (dithiothreitol (DTT)) sex change and non-DTT sex change with immuning hybridization (western blot) method.
Step is as follows: after 100ng (nanogram) recombinant human PlGF albumen (purchasing the Rand DSystemes company in the U.S.) is dissolved in the PBS-0.1%Tween20 liquid that contains 1%BSA, is containing DTT or do not having under the DTT respectively, 94 ℃ hatch 5 minutes after, last sample is to the SDS-PAGE electrophoresis.Again albumen is gone to pvdf membrane behind the electrophoresis.Pvdf membrane adds monoclonal antibody P16 (whole solubility: the 1ug/ml of antibody is dissolved in the PBS-0.1%BSA liquid) after the washing of PBS-0.1%Tween20 liquid, hatched 1 hour for 37 ℃.Behind PBS-0.1%Tween20 liquid thorough washing, add the anti-mouse IgG antibody of horseradish peroxidase (HRP) labelled goat (available from U.S. Sigma company) again, hatched 1 hour for 37 ℃.Behind PBS-0.1%Tween20 liquid thorough washing, add DAB-0.1%H
2O
2Substrate solution colour developing 10-15min is with 0.1M HCl termination reaction.The colour developing result is as shown in Figure 3: wherein band 1 and 2 people PlGF protein sample be for purchasing the R and D Systemes company in the U.S., and band 3 and 4 people PlGF protein sample are for press separation acquisition among the embodiment 1.This experimental result clearlys show: monoclonal antibody P16 of the present invention can with DTT sex change and unmodified people PlGF albumen specific combination.
The external biological activity research of the proteic monoclonal antibody of embodiment 4. anti-people PlGF
In the present embodiment, the P16 monoclonal antibody of the purifying of different solubility (by embodiment 3 preparations) and the fixing biotin labeled recombinant soluble VEGFR1-Fc receptor protein of solubility (available from U.S. Rand D Systmes company) were EP test tube incubated in vitro 5 minutes, again antibody-biotin labeling receptor protein mixture is transferred to and wraps in advance by (bag is 5ug/ml by solubility in the 96-orifice plate of people PlGF albumen (by embodiment 1 preparation), the 50ul/ hole), hatched 1 hour for 37 ℃.After the washing of PBS-0.1%Tween20 liquid, add the Avidin (HRP-Avidin) of horseradish peroxidase (HRP) mark, hatched 1 hour for 37 ℃.Behind PBS-0.1%Tween20 liquid thorough washing, add O-Phenylene Diamine (OPD)-0.1% H
2O
2Substrate solution colour developing 10-15min with the 0.1MHCl termination reaction, reads the OD of 492nm place value (OD at 492nM) in MK3-Multiskan microplate reader (Thermo Scientific company product).Detected result such as Fig. 4.
As shown in Figure 4, after adding the unlabelled P16 monoclonal antibody of biotin labeled VEGFR1-Fc receptor protein and different solubility, the amount relation of being inversely proportional to of the unmarked P16 antibody of its OD value and adding: promptly the amount of the unmarked P16 antibody of Jia Ruing is high more, and its OD value is low more.But this result proves the P16 monoclonal antibody and combines with PlGF is proteic at external specific blockage VEGFR1 acceptor.
The proteic monoclonal antibody of embodiment 5. anti-people PlGF suppresses the growth of people's voluntary muscle transplanted tumor in nude mouse
Step 1: the foundation of people A673 voluntary muscle transplanted tumor model
Collect the A673 rhabdomyoma cell (available from school of life and health sciences cell preservation center, Chinese Academy of Sciences Shanghai) of vitro culture, altogether collecting cell quantity about 10
6/ ml * 8ml, 1000rpm are after centrifugal 4 minutes, and resuspended with 8ml physiological saline, EP manages packing, every pipe 0.2ml, mixing before the injection.With the A673 oncocyte of mixing be inoculated in 8 week Balb/ male nude mouse in age (nude mice is available from Shanghai Slac Experimental Animal Co., Ltd.) right sides subcutaneous.Every nude inoculation 5x10
6Cell is only inoculated 20-30 altogether).Wait to inoculate back 10-14 days, when tangible lump is seen in inoculation place, the treatment of beginning random packet.
Step 2:P16 mab treatment people A673 voluntary muscle transplanted tumor
The above-mentioned nude mice that has inoculated the A673 knurl is divided into four groups (every groups 6-8 only), gives respectively: 1) P16 monoclonal antibody (by embodiment 3 preparations, i.e. P16 Antybody therapy group, 200ug/ time) by abdominal injection; 2) Avastin antibody (200ug/ time) contrast IgG (being IgG antibody control group) and 3) from the discarded residue product after the tumour patient medication of the clinical clinic of New York Maimonides available from U.S. Sigma company, 200ug/ time.Each organizes the first administration time for the 13rd day behind the inoculated tumour cell, after this is administered once every 3 days, amounts to 10 times, to the 43rd day.Measure every group of tumour size in each mouse during this time, observations as shown in Figure 5: compare with contrast IgG group, P16 mab treatment group growth of tumor obtains obvious suppression; Its curative effect that suppresses tumour is not second to Avastin Antybody therapy control group.Therefore, this test result proof P16 monoclonal anti physical efficiency effectively suppresses people A673 tumour in the intravital growth of nude mice.
Being used in combination of the proteic monoclonal antibody of embodiment 6. anti-people PlGF is used for detection by quantitative sample people PlGF albumen.
Step 1: the selection of coated antibody
In the present embodiment, earlier (code name is respectively P5, P21-2 with proteic each monoclonal antibody of anti-people PlGF of purifying, P61, P28 and P38 derive from embodiment 2) be coated antibody, wrap respectively by (bag is 5ug/ml by solubility, the 50ul/ hole) in the 96-orifice plate, 4 ℃ are spent the night.
Step 2: sample is hatched
The 96-orifice plate adds the sample that contains different solubility people PlGF albumen (by embodiment 1 preparation) after 2% bovine serum albumin (BSA) sealing and the washing of PBS-0.1%Tween20 liquid, hatched 2 hours for 37 ℃.
Step 3: detect antibody incubation
Above-mentioned 96-orifice plate adds biotin labeled P16 monoclonal antibody (by embodiment 3 preparations) for detecting antibody after PBS-0.1%Tween20 liquid washing, 37 ℃ hatch 1 hour after; Add the Avidin (HRP-avidin, U.S. Sigma company product) of horseradish peroxidase (HRP) mark again, hatched 1 hour for 37 ℃.
Step 4: substrate colour developing
Above-mentioned 96-orifice plate adds O-Phenylene Diamine (OPD-0.1% H behind PBS-0.1%Tween20 liquid thorough washing
2O
2Substrate solution colour developing 10-15min is with 0.1M HCl termination reaction.In MK3-Multiskan microplate reader (Thermo Scientific company product), read the OD of 492nm place value (OD at 492nM).
Detected result is as shown in Figure 6: in each group of coated antibody, be used in combination people PlGF albumen in the test sample with P61 monoclonal antibody and biotin labeled P16 monoclonal antibody, the detected result that is obtained is best, but when wherein both are used in combination detection by quantitative to sample in the proteic minimum level of people PlGF at 10ng/ml.
Embodiment 7. is a coated antibody with the proteic monoclonal antibody P16 of anti-people PlGF, serves as to detect antibody to be used in combination people PlGF albumen in the test sample with biotin labeled P61 monoclonal antibody.
In the present embodiment, with the proteic monoclonal antibody P16 of anti-people PlGF of purifying (by embodiment 3 preparations) be the coated antibody bag by (wrapping by solubility is 5ug/ml, the 50ul/ hole) in the 96-orifice plate, 4 ℃ are spent the night.After 2% bovine serum albumin (BSA) sealing and the washing of PBS-0.1%Tween20 liquid, add the sample that contains different solubility people PlGF albumen (by embodiment 1 preparation), hatched 2 hours for 37 ℃.After PBS-0.1%Tween20 liquid washing, add biotin labeled P61 monoclonal antibody (deriving from embodiment 2) for detecting antibody, 37 ℃ hatch 1 hour after; Add the Avidin (HRP-avidin, U.S. Sigma company product) of horseradish peroxidase (HRP) mark again, hatched 1 hour for 37 ℃.Behind PBS-0.1% Tween20 liquid thorough washing, add O-Phenylene Diamine (OPD)-0.1% H
2O
2Substrate solution colour developing 10-15min is with 0.1M HCl termination reaction.In MK3-Multiskan microplate reader (Thermo Scientific company product), read the OD of 492nm place value (OD at 492nM).
Detected result is as shown in Figure 7: with the P16 monoclonal antibody is that coated antibody and biotin labeled P61 monoclonal antibody serve as to detect antibody, but both are used in combination people PlGF albumen in the detection by quantitative sample.The people PlGF albumen minimum level that wherein but detection by quantitative arrives in the sample is at 10ng/ml.
The proteic monoclonal antibody of embodiment 8 anti-people PlGF is used for the people PlGF albumen that immunohistochemical methods detects cell and histopathologic slide
Monoclonal antibody of the present invention also is used for the proteic expression of people PlGF that immunohistochemical methods detects cell and various tissue slice samples.
The P16 monoclonal antibody is used for the people PlGF albumen that immunohistochemical methods detects cell and tissue slice, and its step is as follows:
1) gets the section of people's healthy tissues (people's mammary tissue and colon's section, available from the super English in Shaanxi Bioisystech Co., Ltd) or histopathologic slide (human breast carcinoma tissue and colon cancer tissue section, available from the super English in Shaanxi Bioisystech Co., Ltd) in 60 ℃, baked sheet 30 minutes, conventional dewaxing aquation;
2) antigen retrieval: high pressure was repaired antigen 2 minutes, was cooled to room temperature, and PBS washed 5 minutes;
3) dripped Peroxidase Blocking Solution (the peroxidase blocker derives from Dako company) room temperature 30 minutes;
4) dripping 1 resists: detecting antibody is 4 ℃ of refrigerator overnight of P16 monoclonal antibody (by embodiment 3 preparations, whole solubility: the 5ug/ml of antibody is dissolved in the PBS-0.1%BSA liquid);
5) 0.1%Tween-PBS washes 5 minutes * 3 times;
6) the anti-mouse Ig of the rabbit polyclonal antibody (Rabbitanti-mouse Ig derives from U.S. Sigma company) of dropping horseradish peroxidase (HRP) mark, room temperature 30 minutes;
7) 0.1%Tween-PBS washes 5 minutes * 3 times;
8) DAB colour developing, distillation washing color development stopping;
9) Hematorylin is redyed, washes, is broken up the abundant washing in back and returns indigo plant;
10) conventional dehydration is transparent, the neutral gum mounting; In the microscopically observation and the shooting of taking pictures.
The take pictures immunohistochemical methods section of the cell that observes and tissue of microscopically the results are shown in Figure 8.Wherein Fig. 8 A is the Chinese hamster ovary celI of people PlGF gene transfection, Fig. 8 B is the section of people colon, Fig. 8 C is people's stomach-tissue, and Fig. 8 D is a human breast carcinoma tissue, and Fig. 8 E and Fig. 8 F are people's colon cancer tissue immunohistochemical methods detected result synoptic diagram (Fig. 8 F is the synoptic diagram under the powerful microscope).Cell and tissue slice detected result show that the P16 monoclonal antibody can detect the PlGF antigen in normal and the tumor tissues.
Claims (8)
1. the proteic monoclonal antibody of anti-people PlGF is characterized in that, but this monoclonal antibody specific combination people placenta growth factor PlGF and block combining of PlGF and its acceptor.
2. a MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 1 comprises the steps:
Step 1, preparation recombinant human PlGF albumen are immunogen;
Step 2, animal immune:, obtain the high proteic mouse polyclonal antibody of anti-people PlGF of tiring of secretion by the subcutaneous immunity of the mouse of repeated multiple times low dose;
Step 3, picking mouse are therefrom got its spleen cell, merge with murine myeloma cell by external;
Step 4, enzyme linked immunosorbent assay screening antibody-secreting male hybridoma;
Step 5, positive hybridoma cell are identified the hybridoma that obtains the proteic monoclonal antibody of the anti-people PlGF of stably excreting through subclone;
Step 6, hybrid tumor cell amplification is cultivated, collected nutrient solution, adopt the affinity chromatography separation and purification to obtain the proteic monoclonal antibody of anti-people PlGF.
3. preparation method as claimed in claim 2, it is characterized in that, described step 1 is specially: pcr amplification obtain the encoding gene segment of people PlGF, it is cloned in the Yeast expression carrier pPic9K carrier, obtain expression plasmid pPic9K-PlGF, transform Pichia yeast respectively, filter out efficient expression engineering strain, engineering strain obtains the recombinant human PlGF albumen of purity more than 95% after fermentation, abduction delivering, separation and purification.
4. preparation method as claimed in claim 2 is characterized in that, the method for the subcutaneous immunity of mouse described in the step 2 is: the people PlGF albumen of described purifying is mixed with Freund's complete adjuvant, in subcutaneous multi-point injection mouse.
5. preparation method as claimed in claim 2, it is characterized in that the described affinity chromatography of step 6 is specially: the hybridoma filtrate supernatant that will contain monoclonal antibody is splined in the proteic 4% agarose particulate pearl affinity chromatographic column of reorganization protein-A that has been pre-charged with covalent cross-linking; After last sample finished, affinity chromatographic column was removed foreign protein with the PBS wash-out, again the antibody protein that is adsorbed by the particulate pearl with glycine liquid wash-out, dialysis again; Dialyzed sample promptly obtains the proteic monoclonal antibody of anti-people PlGF of purifying more after filtering.
6. the described monoclonal antibody of claim 1 application in the people PlGF albumen in the detection by quantitative biological sample.
7. the described monoclonal antibody of claim 1 application in the people PlGF albumen in the qualitative detection biological sample.
8. the application of the described monoclonal antibody of claim 1 in the medicine of preparation treatment tumour.
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| CN2010101480883A CN102219854A (en) | 2010-04-15 | 2010-04-15 | Monoclonal antibody of anti-human PIGF (placental growth factor) protein as well as preparation method and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103207274A (en) * | 2013-04-28 | 2013-07-17 | 成都中医药大学 | Screening kit for seventh chromosome abnormality diseases of fetus |
| CN108314701A (en) * | 2018-04-09 | 2018-07-24 | 青岛汉德森生物科技有限公司 | A method of utilizing magnetic bead extraction and antibody purification |
| CN109134650A (en) * | 2018-09-10 | 2019-01-04 | 宁波奥丞生物科技有限公司 | The preparation method of anti-human PLGF monoclonal antibody |
| CN112358546A (en) * | 2020-11-10 | 2021-02-12 | 宁波奥丞生物科技有限公司 | Hybridoma cell strain 9C1, PLGF-1 monoclonal antibody, preparation method and application thereof |
| CN113801226A (en) * | 2021-11-19 | 2021-12-17 | 南京佰抗生物科技有限公司 | Anti-human PlGF (platelet-derived growth factor) mouse-derived monoclonal antibody and application thereof |
| WO2022099465A1 (en) * | 2020-11-10 | 2022-05-19 | 宁波奥丞生物科技有限公司 | Hybridoma cell strain 9c1, plgf-1 monoclonal antibody, preparation method therefor and application thereof |
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2010
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103207274A (en) * | 2013-04-28 | 2013-07-17 | 成都中医药大学 | Screening kit for seventh chromosome abnormality diseases of fetus |
| CN108314701A (en) * | 2018-04-09 | 2018-07-24 | 青岛汉德森生物科技有限公司 | A method of utilizing magnetic bead extraction and antibody purification |
| CN109134650A (en) * | 2018-09-10 | 2019-01-04 | 宁波奥丞生物科技有限公司 | The preparation method of anti-human PLGF monoclonal antibody |
| CN112358546A (en) * | 2020-11-10 | 2021-02-12 | 宁波奥丞生物科技有限公司 | Hybridoma cell strain 9C1, PLGF-1 monoclonal antibody, preparation method and application thereof |
| CN112358546B (en) * | 2020-11-10 | 2022-04-01 | 宁波奥丞生物科技有限公司 | Hybridoma cell strain 9C1, PLGF-1 monoclonal antibody and application thereof |
| WO2022099465A1 (en) * | 2020-11-10 | 2022-05-19 | 宁波奥丞生物科技有限公司 | Hybridoma cell strain 9c1, plgf-1 monoclonal antibody, preparation method therefor and application thereof |
| GB2616177A (en) * | 2020-11-10 | 2023-08-30 | Ningbo Aucheer Biotechnology Co Ltd | Hybridoma cell strain 9C1, PLGF-1 monoclonal antibody, preparation method therefor and application thereof |
| CN113801226A (en) * | 2021-11-19 | 2021-12-17 | 南京佰抗生物科技有限公司 | Anti-human PlGF (platelet-derived growth factor) mouse-derived monoclonal antibody and application thereof |
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