WO2010063138A1

WO2010063138A1 - A monoclonal antibody specifically binding to vegf and the hybridoma secreting same and uses thereof

Info

Publication number: WO2010063138A1
Application number: PCT/CN2008/001954
Authority: WO
Inventors: 周群敏; 胡红群; 孙亚男; 罗师平; 周青; 徐一清
Original assignee: 苏州思坦维生物技术有限公司
Priority date: 2008-12-01
Filing date: 2008-12-01
Publication date: 2010-06-10
Also published as: WO2010063138A8; CN102124100A; CN102124100B

Abstract

A monoclonal antibody specifically binding to human vascular endothelial growth factor (VEGF) and the hybridoma cell line secreting the monoclonal antibody. The hybridoma cell line is deposited as CGMCC No. 2743 in the Center for General Microbiological Culture Collection, China Committee for Culture Collection of Microorganisms (CGMCC). A method of preparing the monoclonal antibody and the monoclonal antibody chemically labeled in vitro. And the uses of the monoclonal antibody (labeled or unlabeled) in detecting or separating VEGF protein.

Description

Monoclonal antibody specifically binding to VEGF and hybridoma cell line secreting same and use thereof

Technical field:

The invention belongs to the field of biotechnology-monoclonal antibodies. The present invention relates to a monoclonal antibody which specifically binds to human VEGF and a hybridoma cell line secreting same and use thereof.

Background technique:

Angiogenesis or angiogenesis biologically refers to the process by which existing blood vessels (such as capillaries and venules) in the body produce new blood vessels by budding or dividing. Angiogenesis is beneficial and necessary to maintain many of the normal physiological processes of the body, such as tissue embryo development, healing and repair of traumatic wounds. However, excessive angiogenesis or hyperplasia is also closely related to many pathological changes (such as tumor hyperplasia, inflammation).

The key to the angiogenesis or proliferation of blood vessels in the body is that the vascular endothelial cells lining it have the ability to divide and directionally migrate into the existing vessel wall. Among the various factors that stimulate vascular endothelial cell proliferation, vascular endothelial growth factor (VEGF) is particularly important. In fact, VEGF is currently the most potent factor in stimulating vascular endothelial cell division and proliferation. The importance of VEGF in stimulating angiogenesis has also been confirmed in studies of VEGF knockout mice: as long as one of the VEGF genes is knocked out, the blood vessels in the embryo cannot form normally and the embryo is developing. It dies by 11 to 12 days (Carmel iet P et al. Nature 1996, 380: 435; Ferrara N et al. Nature 1996, 380: 439).

VEGF is commonly synthesized by vascular endothelial cells, macrophages, and tumor cells, and acts specifically on VEGF receptors on vascular endothelial cells through autocrine/paracrine methods to promote vascular endothelial cell growth, proliferation, and migration. , formation and other functions. Recent studies have shown that VEGF can also be detected in cancer tissues of most malignant tumor patients, and that increased expression of VEGF is associated with tumor malignancy and disease progression (Dvorak HF et al: J Exp Med 1991 : 174 : 1275-8 ; Brown LF et al. Cancer Res 1993; 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 is in the blood vessels of inflammatory pathology Play an important role, such as rheumatoid synovial fluids and tissue in arthritic patients And increased levels of VEGF in tissues of patients with psoriasis (psoriasis). Blocking VEGF in the body, thereby preventing angiogenesis induced by VEGF and its receptor, can inhibit tumor growth and metastasis, or anti-inflammatory effects.

There are two main methods in the field of drug research and development that inhibit VEGF and its receptor-mediated angiogenesis: namely:

1) Search for substances that directly inhibit the intrinsic tyrosine protein kinase activity in VEGF receptors. The drugs developed in this class are mainly small molecule inhibitors such as PTK787/ZK 222584 (Wood JM et al. Cancer Res 2000, 60: 2178);

2) By inhibiting the binding of VEGF to its receptor, thereby preventing VEGF and its receptor-mediated pro-angiogenic effects, such drugs include various anti-VEGF or anti-VEGF receptor antibodies, antisense oligo Glycoside and small molecule inhibitors, etc. (Kim KJ et al. Nature 1993, 362: 841; Presta LG et al. Cancer Res, 1997, 57: 4593; Posey JA et al. Clinical Cancer Research, 2003, 9: 1323). Bevacizumab (trade name Avastin), which was developed and produced by Genentech in the United States and approved for sale by the US FDA in 2004, is a humanized monoclonal antibody that recognizes and binds to VEGF. Avastin inhibits tumor angiogenesis by neutralizing or eliminating VEGF in the body, thereby inhibiting tumor growth and metastasis (Presta LG et al. Cancer Res, 1997, 57: 4593; Hurwitz H et al. N Engl J Med, 2004: 350: 2335 ).

The precursor of the humanized antibody Avastin can be traced back to the mouse monoclonal antibody code-named A4.6.1. The source of the A4.6.1 mouse monoclonal antibody and the hybridoma cell line secreting the same are disclosed in US Patent No. 6,582,959 (Inventor: ! (^, 1^111^ ₁ 1 , Patent Disclosure) Date: June 24, 2003, patent name: Antibodies to vascular endothelial cell growth factor); and US Patent No. 7,227,004 (inventor: 1^111, 1^1^ _§) Patent publication date: June 5, 2007, patent name: Antibodies to vascular endothelial cell growth factor) are described. However, the antibody also has the following disadvantages: (1) Like most monoclonal antibodies, A4.6.1 mouse monoclonal antibody and its human The activating antibody Avastin is only a part of the site that binds to the VEGF antigen and cannot recognize or cover many other antigenic sites on VEGF. (2) Further animal experiments and clinical studies have found that only A4.6.1 mouse monoclonal antibody is injected alone. Or its humanized antibody Avastin, unable to achieve complete neutralization inhibits VEGF and its mediated angiogenesis in vivo.

Therefore, the development of more new monoclonal antibodies against human VEGF and the hybridoma cell line secreting it are obvious. It makes sense and necessary.

Summary of the invention:

It is an object of the present invention to provide monoclonal antibodies which specifically bind to native VEGF or denatured VEGF.

Another object of the present invention is to provide a hybridoma cell line secreted to produce the above monoclonal antibody. Still another object of the present invention is to provide a process for the preparation of the monoclonal antibody of the present invention.

Another object of the invention is to provide an in vitro chemically labeled monoclonal antibody.

Another object of the present invention is to provide a monoclonal antibody of the present invention for use in detecting a VEGF protein.

Another object of the invention is to provide the use of in vitro chemically labeled monoclonal antibodies for the detection of VEGF proteins.

Another object of the present invention is to provide an application of the monoclonal antibody of the present invention for isolating a VEGF protein. .

The invention selects recombinant human VEGF protein as an immunogen, and obtains a high-valence anti-VEGF polyclonal antibody by repeatedly subcutaneously immunizing a small dose of mice; and then picking up the mouse and taking the spleen cells thereof, through in vitro and small Hybridoma monoclonal cells stably secreting anti-human VEGF antibody were established by fusion of murine myeloma cells, drug screening and subcloning. One of the strains, M23 hybridoma cell line, was identified by ELISA, immunoblotting, immunohistochemistry, etc., and it was confirmed that the secreted monoclonal antibody can specifically recognize and bind human VEGF (including VEGF121, VEGF165 and VEGF189). In vitro studies have also demonstrated that this monoclonal antibody can compete to inhibit the binding of the VEGF receptor to its ligand (i.e., VEGF). In one aspect of the invention, a hybridoma cell line capable of secreting a monoclonal antibody, which is coded M23, has been deposited with the General Microbiology Center of the Chinese Collection of Microorganisms Collections on November 14, 2008. (referred to as CGMCC), the deposit number is CGMCC No. 2743, and the deposit location is: China, Beijing.

In another aspect of the present invention, a monoclonal antibody which specifically binds to a VEGF protein is produced and secreted by the hybridoma cell line having the above-mentioned accession number CGMCC No. 2743.

In another aspect of the present invention, a method for preparing the above monoclonal antibody, comprising the steps of: Step 1. Prepare recombinant human VEGF protein as immunogen; Step 2. Animal immunization: Obtain high titer anti-human VEGF polyclonal antibody by repeated subcutaneous immunization of small doses of mice; Step 3: Pick mice from The spleen cells are obtained and fused with mouse myeloma cells in vitro: Step 4. The antibody-positive hybridoma cells are screened by enzyme-linked immunosorbent assay; Step 5: The positive hybridoma cells are subcloned to obtain multiple stable secretion resistance Hybridoma monoclonal cells of human VEGF antibody; Step 6. The hybridoma cells are expanded and cultured, the culture solution is collected, and the purified anti-human VEGF monoclonal antibody is separated by affinity chromatography.

Wherein, the step 1 is specifically: PCR-amplifying a gene fragment encoding human VEGF, and cloning it into the yeast expression vector pPi9K vector, obtaining the expression plasmid pPi9K-VEGF, transforming the yeast, screening the high-efficiency expression engineering strain, and engineering the strain After fermentation, induced expression, isolation and purification, VEGF protein with a purity of more than 95% is obtained.

The method for subcutaneous immunization of the mouse in the step 2 is: mixing the purified recombinant human VEGF protein with Freund's complete adjuvant, and injecting the mouse into the skin at a multiple point.

The affinity chromatography method described in the step 6 is specifically: loading the supernatant of the hybridoma cell containing the monoclonal antibody onto the microparticle bead affinity chromatography column pre-filled with the recombinantly cross-linked recombinant human VEGF protein. After the sample is completed, the affinity column is eluted with PBS to remove the heteroprotein, and the antibody protein adsorbed by the microparticle beads is eluted with glycine solution, and then dialyzed; the dialyzed sample is filtered to obtain the purified anti-VEGF. Monoclonal antibodies.

In another aspect of the invention, there is provided an in vitro chemically labeled monoclonal antibody as described above, the labeling steps of which are as follows:

1), dissolving the above monoclonal antibody in PBS;

2), adding an appropriate amount of labeling reagent and mixing with the monoclonal antibody sample, and mixing, and reacting at room temperature;

3), the unreacted excess labeling reagent is removed by dialysis or desalting, and the monoclonal antibody chemically labeled in vitro is obtained.

In another aspect of the invention, there is provided a use of the above monoclonal antibody for detecting a VEGF protein. The present invention further confirmed that the above monoclonal antibody is used for detecting the expression of VEGF protein in body fluids or diseased tissues. For example, the monoclonal antibody is used as a reagent for quantitatively detecting the level of VEGF in various body fluids such as blood, plasma, serum, urine, lymph or cerebrospinal fluid, or detecting various biological materials such as cell culture supernatants and tissue cells. The level of VEGF in the lysate; or the monoclonal antibody as a reagent for immunohistochemistry or the like to qualitatively or quantitatively detect the expression of VEGF protein in various diseased organs, or various diseases Under the disease, the expression of VEGF in the peripheral blood of patients with tumor (early stage of tumor hyperplasia and diffusion), inflammation (such as arthritis, systemic red wolf larvae, etc.) is used as a marker for clinical diagnosis or auxiliary diagnosis.

In another aspect of the invention, there is provided a use of the above-described in vitro chemically labeled monoclonal antibody for detecting a VEGF protein. Labeled (such as biotin, radiosin or fluorescein such as FITC) anti-VEGF monoclonal antibody is a probe developer for immunofluorescence experiments, autoradiography or other development methods to locate or quantify various diseases VEGF expression levels in tissues and organs such as tumor or inflammatory patients to aid clinical diagnosis.

In another aspect of the invention, there is provided the use of a monoclonal antibody as described above for isolating a VEGF protein. The monoclonal antibody is used to prepare an immunoaffinity chromatography column to separate and purify the VEGF protein.

The monoclonal antibody of the present invention can also be used as an antagonist of VEGF for basic medical research such as pharmacology. Further, the M23 hybridoma cell line secreting the antibody can be used to isolate and purify mRNA, and the gene encoding the light chain and heavy chain variable regions of the M23 antibody can be cloned and amplified by RT-PCR or the like. The obtained antibody light chain and heavy chain variable region genes can be used to prepare various genetically engineered antibodies such as single-chain antibodies, Fab fragments, human-murine antibodies or humanized antibodies. M23 antibodies or derivatives thereof (such as antibody fragments, chimeric antibodies, humanized antibodies, radiopharmaceutically labeled antibodies, etc.) can be used as a separate component or in combination with other drugs to prepare lesions associated with high expression of VEGF (eg, tumors). Therapeutic drugs or preparations for proliferation, inflammation, etc.). BRIEF DESCRIPTION OF THE DRAWINGS:

1 is a schematic diagram showing the results of an experiment in which a monoclonal antibody having high affinity binding to a recombinant VEGF165 protein (m23) in a culture supernatant of M23 hybridoma cells was confirmed by ELISA in Example 1, wherein X653 represents unfused. P3X63. Ag8. 653 myeloma cell culture supernatant was used as a negative control.

Fig. 2 is a schematic diagram showing the results of electrophoresis experiments of M23 antibody protein purified by VEGF affinity chromatography column under the conditions of DTT reduction by SDS-PAGE in Example 2. Lanes 1, 2, 3, and 4 represent four parallel loadings of the same batch of products (the amount of protein loaded per lane is the same, 5 y g). The M23 antibody protein was derived from the culture supernatant of the Μ23 hybridoma cells.

3 is a schematic diagram showing the results of an experiment for detecting the binding reaction of monoclonal antibodies secreted by M23 hybridoma cells to recombinant VEGF165 protein (reduced and unreduced) by Western-blot analysis in Example 3. Lane 1 (unreduced VEGF165 protein) and lane 2 (reduced VEGF165 protein) For the results of detection with the M23 monoclonal antibody; Lane 3 (unreduced VEGF165 protein) and Lane 4 (reduced VEGF165 protein) are the results of detection with rabbit polyclonal antibody.

Fig. 4 is a schematic diagram showing the results of the specificity of the reaction of the M23 monoclonal antibody by ELISA in Example 4. The results showed that the M23 monoclonal antibody binds to the VEGF165 protein but does not bind to the placental growth factor (PLGF) protein.

5 is a schematic diagram showing the expression of VEGF protein (VEGF12K VEGF165 and VEGF189) in CH0 (Chinese hamster ovary cells) cells by immunohistochemistry using the purified M23 monoclonal antibody as a reagent component in Example 5; For transfection of cells containing the VEGF121 gene plasmid, Figure 5B is a cell transfected with a plasmid containing the VEGF165 gene; Figure 5C is a cell transfected with a plasmid containing the VEGF189 gene; Figure 5D is a cell transfected with a blank control plasmid.

Figure 6 is a comparison of biotin-labeled M23 monoclonal antibody (Biotin-m23, initial dilution solubility: 8000) in Example 6 to detect antibody dilution of VEGF coated on 96-well microtiter plate. Degree - response curve.

Figure 7 is a graphical representation of the results of an experimental ELISA assay for the competitive binding of biotinylated M23 monoclonal antibody to VEGF receptor protein (F2K3FC) in combination with VEGF in Example 7. The PLGF protein is a negative control.

Fig. 8 is a schematic view showing the results of an experiment for analyzing VEGF protein in human pathological tissue sections by immunohistochemistry using M23 monoclonal antibody as a reagent in Example 8. Figure 8A is a human gastric adenocarcinoma tissue section; Figure 8B is a human lymph node tissue section. The best way to implement the invention:

The following examples are intended to provide a more complete understanding of the invention, and are not intended to limit the invention.

Example 1: Establishment and Screening of Hybridoma Cell Line Stabilizing Secretion of Anti-VEGF Monoclonal Antibody Step 1. Preparation of Recombinant Human VEGF165 Protein (Immune Antigen) (Application No. 2007100473522, entitled "Recombinant Human Vascular Endothelial Cells" Isolation and purification of growth factors, in vitro chemical labeling and its application are prepared by the method described in the Chinese invention patent application:

A gene fragment encoding human VEGF165 open-reading frame (ORF) was amplified from human lung tissue cell cDNA library by PCR and identified by sequence analysis. Indeed, after treatment with restriction endonucleases, they were cloned into the yeast expression vector pPi9K (Invitrogen) vector. The expression plasmid _P Pi9K-VEGF165 was obtained, transformed into Pichia pastoris, and the highly efficient engineered strain was screened. The engineered bacteria are fermented and induced to express, and after isolation and purification, VEGF165 protein with a purity of more than 90% is obtained.

Step 2. Animal immunization:

The purified recombinant human VEGF165 protein was mixed with Freund's complete adjuvant, and C57BL/6 mice (moving 1/10, a total of 10 ug of VEGF165 protein) were injected subcutaneously. After the first immunization for 2 to 3 weeks, the mice were given a subcutaneous injection of VEGF165 protein and an incomplete adjuvant mixture to boost the immunization. After boosting the immunization for 2 to 3 times, a small amount of mouse serum was taken, and the titer of the anti-VEGF protein antibody in the serum of the mouse was detected by ELISA using a 96-well microtiter plate coated with VEGF165 protein, and the mouse spleen cell with high titer was used. Cell fusion for the next step.

Step 3: Cell fusion - 3 days after the last immunization of VEGF165 protein, the mouse spleen cell suspension was prepared aseptically, and P3X63. Ag8.653 mouse myeloma cells (purchased from the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences) The ratio of 1 is fused by 50% PEG-1500 (product of Sigma, USA). Fusion according to the conventional method (ohler G. and Mi lstein C: Nature 1975; 256: 495-497), the amount of PEG lml, slowly added within 1 minute. After 90 seconds of reaction, the reaction was stopped in serum-free RPMI-1640 medium, centrifuged at 100 rpm for 10 min, the supernatant was removed, and the cells under centrifugation were centrifuged to contain 10% HAT (H is hypoxanthine, sputum amino oxime) , T thymidine, Sigma product RPMI 1640-10% FCS medium to adjust the cell concentration to l X 107ml, add 96-well flat-bottomed cell culture plate (200ul per well), at 37 ° C, 5% Incubate for 2-3 weeks in a C02 incubator.

Step 4. Enzyme-linked immunosorbent assay (ELISA) for screening antibody-positive hybridoma cells: coated with recombinant human VEGF165 protein (5 μg/ml, pH 9. 6, 0.1 M NaHC03 solution), Coating at 37 ° C for 2 hours or 4 ° C overnight; 2% bovine serum albumin (BSA) blocked, overnight at 4 ° C. After washing with PBS-0. 1% Tween20 solution, the culture supernatant of the hybridoma cells to be detected was added (unfused P3X63. Ag8. 653 myeloma fine culture supernatant was used as a negative control) and incubated at 37 °C for 2 hours; After washing with 0% 1% Tween20 solution, horseradish peroxidase (HRP)-labeled goat anti-mouse Ig (Sigma product) was added and incubated at 37 °C for 1 hour; then PBS-0. 1% Tween20 solution was fully After washing, o-phenylenediamine (0PD) -0. 1% 0 ₂ substrate solution was developed for 10-15 min, and the reaction was terminated with 0.1 M HCl. The 0D value at 492 nm was read in a MK3 Multiskan microplate reader (product of Thermo Scientific). Measured The hybridoma cells having a 0D 492 value 5-10 times higher than the negative control were re-cloned and expanded for cryopreservation. Step 5. Cloning of positive hybridoma cells - limiting dilution method

The positive cells obtained by the above screening were diluted to 1-10 cells per well in RPMI-1640-10% FCS medium, placed in a 96-well cell culture plate, and cultured in a 37 V, 5% CO 2 incubator. 3 weeks. After the clone was grown, the supernatant was taken for re-testing to identify the secretion of anti-VEGF antibody. After detection and identification, a plurality of antibody-secreting positive cell lines were obtained. Among them, after further subcloning and identification, a hybridoma cell line stably secreting anti-VEGF monoclonal antibody with the code of M23 was obtained. Figure 1 shows the binding of the supernatant of M23 hybridoma cells to the recombinant VEGF165 protein by ELISA. The results showed that the supernatant of the hybridoma cells contained a monoclonal antibody with high titer against VEGF165 protein. This monoclonal antibody was identified as an IgG1 class. The hybridoma cell line was further expanded, long-term subcultured and cryopreserved. It was preserved at the General Microbiology Center of the China Microbial Culture Collection Management Committee on November 14, 2008, and the accession number was CGMCC NO. 2743.

Example 2. Preparation and purification of anti-VEGF monoclonal antibody in vitro

The established M23 hybridoma cells were expanded and cultured in a serum-free medium, and the dosing was stopped when the cell concentration reached 10 ⁵ /ml or more, and the culture was continued until the cell culture solution turned yellow. The culture solution was collected, centrifuged at 1500 rpm for 10 minutes, and the supernatant was filtered through a 0.45 μηη filter and stored at 4 ° C or -20 Torr for isolation or purification of the monoclonal antibody directly used in the next step.

In this example, the separation and purification of the anti-VEGF monoclonal antibody (M23) was carried out by affinity chromatography. The purification step is: loading the supernatant of the hybridoma cell containing M23 monoclonal antibody onto 4% agarose beads (agarose beads) pre-filled with recombinant human VEGF165 protein (prepared by Example 1). , Sigma (American Sigma) affinity column (lL / lOml column); After loading, the affinity column is eluted with PBS to remove the heteroprotein, followed by low pH (2.7) glycine ( 0. 1M) The liquid elutes the adsorbed antibody protein. The eluate was adjusted to pH 7.0 with 1 mol/L Tris (pH 9.0), and then dialyzed against 10 times volume of lx PBS for 12 to 16 hours (after changing 2-3 times), after dialysis The purified anti-VEGF monoclonal antibody was obtained by filtering the sample through a 0.45 μm filter.

The purified antibody was identified by SDS-PAGE electrophoresis (10% separation gel, 5% concentrated gel) by a conventional method. Figure 2 is the results of SDS-PAGE electrophoresis under DTT reducing conditions, wherein the bands above represent the M23 antibody heavy chain and the lower bands represent the M23 antibody light chain. Example 3. Western blot Analysis of the binding of M23 monoclonal antibody to recombinant human VEGF165 protein

Figure 3 shows the results of immunoblot (Western blot) assay for the specific binding reaction of purified M23 monoclonal antibody to human VEGF165 protein. The VEGF protein used in this example to identify the specificity of the M23 monoclonal antibody was derived from recombinant human VEGF165 protein expressed in E. coli (purchased from R and D Systems, USA). The VEGF165 protein was first dissolved in PBS-1% BSA solution (50 μg/ml), and each sample was loaded with 10 μl for 15% SDS-PAGA electrophoresis, and the gelatin band was transferred to the PVDF membrane by a conventional method (Mil Lipore company), blocked with 5% skim milk powder at 4 °C overnight, added M23 monoclonal antibody prepared in Example 2, and added rabbit anti-human VEGF polyclonal antibody (product of Santa Cruz Biotechnology, USA) to the positive control. Hour, washed 3 times with TBS-T (pH 7.5, 0.05 mol/l Tris-Hcl, 0.15 mol/l NaCl, 0.05% Tween-20), followed by horseradish peroxidase (HRP) The labeled goat anti-mouse IgG or goat anti-rabbit IgG (product of Sigma) was reacted at room temperature for 1 h and washed three times with TBS-T. Finally, add DAB- 0. 1% 0 ₂ substrate liquid for 10-15min. As a result, as shown in Fig. 3, M23 monoclonal antibody had a strong specific reaction with reduced (lane 1) and unreduced recombinant VEGF165 protein (lane 2), but did not cross-react with other proteins such as BSA in the lane. The results of this immunoblotting were consistent with the results of the positive control using rabbit anti-human VEGF polyclonal antibody (lane 3 and lane 4).

Therefore, in line with the ELISA results, the results of Western blot showed that the monoclonal antibody produced by the M23 cell line can bind to the human VEGF165 protein.

Example 4. Determination of reaction specificity of M23 monoclonal antibody

In view of the fact that human placental growth factor (PLGF) protein is homologous to human VEGF165 protein, and both have more than 50% identical sequences; in this example, recombinant human VEGF165 protein was separately coated (prepared in Example 1). Or 96-well plates of recombinant PLGF protein (purchased from R and D Systems, USA), using ELISA to identify the reaction specificity of the M23 monoclonal antibody. The ELISA test procedure was the same as in Example 2. The results of the ELISA assay are shown in Figure 4. In the wells coated with VEGF165 protein (5 μg/ml, 50 μl/well), Μ23 monoclonal antibody and HRP-labeled goat anti-mouse Ig and substrate were added. The color reaction was significantly positive, and the color intensity (0D 492 value) was significantly positively correlated with the solubility of the M23 monoclonal antibody added to the well; while the same amount was coated (5 μg/ml, 50 μΐ/well). PLGF protein Sample group, no obvious color development. This result demonstrates that the M23 monoclonal antibody binds to the VEGF protein but does not bind to the PLGF protein.

Example 5. Immunohistochemistry detection of M23 monoclonal antibody binding to VEGF121, VEGF165 and VEGF189 proteins

In this example, the M23 monoclonal antibody was used as a primary antibody reagent for immunohistochemistry to detect various VEGF proteins (such as VEGF121, 165 and 189, etc.) expressed in the transfected cells. To this end, the CH0 cells in the logarithmic growth phase were routinely digested with trypsin/5 mM EDTA, and then inoculated into a 24-well culture plate at 1×10 ⁵ cells/well, and cultured overnight at 37° C., 5% CO 2 ; 2O fugen6 (purchased from Roche-Shanghai) and plasmid DNA containing recombinant human VEGP gene (VEGF121, VEGF165 and VEGF189) or control empty vector plasmid DNA (1-2 ug) in 100 μL serum-free, antibiotic-free DMEM The mixture was mixed and prepared into fugen6-DNA mixture. After being placed at room temperature for 15 min, it was added dropwise to CH0 cells. After transfection for 48 hours, CH0 cells were rinsed with lx PBS solution and fixed at 90% methanol 4 V for 20 min. M23 monoclonal antibody (1: 100 dilution, 200 ul/well), 37 1 h, eluted with PBS, then horseradish peroxidase-labeled goat anti-mouse Ig (1:200 dilution, 200 ul/) Hole), 37 °C for 1 h, eluted with PBS, then added color solution (DAB- 3% H ₂ 0 ₂ ) at room temperature for 5-10 min. The reaction was terminated by rinsing with PBS, and the color reaction was observed under a microscope. The results are shown in Figure 5. In the sample wells transfected with VEGF121 (Fig. 5A), VEGF165 (Fig. 5B) and VEGF 189 (Fig. 5C) expression plasmids, nearly 5-10% of the cells were positive for coloration, while transfection was The samples of the empty vector control (Fig. 5D) showed no color positive cells. The results of this experiment demonstrate that the M23 monoclonal antibody not only recognizes the VEGF165 protein used for immunization, but also recognizes VEGF121 and VEGF189 proteins.

Example 6 In Vitro Biotin Labeling of M23 Monoclonal Antibody

In this example, N-hydroxysuccinimide (NHS) ester-activated biotin (Biotin) (purchased from Pierce, USA) was used as a chemical labeling reagent to label the M23 monoclonal antibody protein. In pH 7-9 buffer, NHS-activated biotin reacts very efficiently with the primary amino group of the protein (-NH2) to form a stable amide bond. In this embodiment, the molar ratio of the biotin labeling reagent to the M23 monoclonal antibody protein is 50:1; the labeling steps are as follows:

1), dissolving the M23 monoclonal antibody in PBS; 2), according to the above calculation results, add appropriate amount of labeling reagent (NHS-biotin) and M23 monoclonal antibody sample mixed, and mix; room temperature reaction for 30-60 minutes;

3), removing unreacted excess biotin by dialysis or desalting to obtain biotin-labeled M23 monoclonal antibody (Biotin-M23).

The biotinylated M23 monoclonal antibody (Biotin-M23) can be used in combination with horseradish peroxidase or fluorescently labeled Avidin to form a kit for qualitative or quantitative detection of VEGF protein. Figure 6 shows the enzyme-labeled human VEGF165 protein (5 wg/ml, 50 ul/well) with biotinylated M23 monoclonal antibody (Biotin-M23, initial dilution 1: 8000, ) An antibody, a dilution-response curve measured by horseradish peroxidase-labeled Avidin (purchased from Vector Laboratories, USA, dilution 1:5000) as a detection reagent. The results indicated that the biotinylated M23 antibody also retained high affinity binding to the VEGF protein at very high dilutions (1: 8000 to 1:80000).

Example 7 Competitive ELISA assay M23 monoclonal antibody competes with VEGF receptor for binding VEGF M23 monoclonal antibody competes with VEGF receptor protein for binding VEGF assay using competitive ELISA.

The competitive ELISA assay steps are as follows:

1) Coating 96-wel l plates (2 μg/ml, 50 μl/well) with recombinant human VEGF165 protein, 4 ° overnight;

2) After rinsing with PBS solution and blocking with 2% BSA (in PBS-0. 1% tween20 solution) at room temperature, add biotin-labeled M23 monoclonal antibody (Bio_M23) with fixed solubility (1: 8000) and different dissolution. Degree of VEGF receptor protein (the receptor protein is F2K3Fc, which is a fusion protein linked to the Fc segment of human immunoglobulin by the extracellular domain of VEGF receptor, using the application number 20081001915922, the invented name is "combinable vascular endothelium Recombinant protein of cell growth factor and placental growth factor, and preparation method and application thereof are prepared by the method described in the Chinese invention patent application; or biotin-labeled M23 monoclonal antibody with fixed solubility (1: 8000) (Bio-M23) ) with different solubility control proteins (PLGF), incubated at 37 °C for 2 h _;

3) After elution with PBS-T, horseradish peroxidase-labeled (1:5000) was added and incubated at 37 °C for 1 h _;

4) After elution with PBS-T, add coloring solution (o-phenylenediamine) -3% hydrogen peroxide, room temperature for 10 min, until Color development

5) The reaction was terminated by the addition of HCL, and the absorbance of each well at a wavelength of 492 nm was measured by an enzyme-linked immunosorbent assay. The results of the competitive ELISA are shown in Figure 7: in the sample group of biotinylated M23 monoclonal antibody with different solubility of unlabeled VEGF receptor protein (ie F2K3Fc & Bio-M23), the 0D value and the added The amount of unlabeled VEGF receptor protein is inversely proportional: that is, the higher the amount of unlabeled VEGF receptor protein added, the lower the OD value. In the sample group of biotin-labeled M23 monoclonal antibody and unlabeled PLGF protein (ie, PLGF & Bio_M23) with different solubility, the 0D value had little relationship with the amount of unlabeled PLGF protein added. This result indicates that the M23 monoclonal antibody competes with the VEGF receptor protein for binding to VEGF.

Example 8 23 Monoclonal antibody for immunohistochemistry Detection of VEGF in pathological tissue sections The M23 monoclonal antibody of the present invention was also used for immunohistochemical detection of VEGF protein in various pathological tissue section samples.

The steps of immunohistochemical detection of VEGF protein in pathological tissue sections are as follows:

1) Pathological tissue section (purchased from Shaanxi Xi'an Chaoying Biotechnology Co., Ltd.) baked in 60 30 for 30 minutes, conventional dewaxing hydration

2) Antigen retrieval: High pressure repair antigen for 2 minutes, cool to room temperature, wash in PBS for 5 minutes X 2 times;

3) Add Peroxidase Blocking Solution at room temperature for 30 minutes;

4) Add 1 anti-antibody: detection antibody M23 monoclonal antibody and negative control antibody (both 1: 10 dilution) 4 °C refrigerator overnight;

5) 0. l%Tween - wash in PBS for 5 minutes X 3 times;

6) Add horseradish peroxidase (HRP)-labeled goat anti-mouse Ig polyclonal antibody at room temperature for 30 minutes;

7) 0. l% Tween-PBS wash 5 minutes X 3 times;

8) DAB color development, distilled water wash to stop color development;

9) After hematoxylin counterstaining, washing, and differentiation, fully washed and returned to blue;

10) Conventional dehydration and transparent, neutral gum seals, observed under microscope and recorded results. Figure 8 is a photograph of a partial pathological tissue section test result. Figure 8A shows a human gastric adenocarcinoma tissue section; it is a human lymph node tissue section; the results indicate that the expression of VEGF protein is detected in both tissue sections.

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International Application No. PCT/CN 2008/ 00 1 954 Notes on the preservation of microorganisms

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Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing, China, Post Code: 100101 Date of Deposit November 14, 2008 Deposit No. CGMCC No.2743

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Claims

Claim

A hybridoma cell line capable of secreting a monoclonal antibody, the preservation number of which is CGrtCC No. 2743.

A monoclonal antibody which specifically binds to VEGF, which is secreted by the hybridoma cell line of claim 1.

3. A method for preparing a monoclonal antibody according to claim 2, comprising the steps of: Step 1, preparing a recombinant human VEGF protein as an immunogen; Step 2, animal immunization: subcutaneous immunization by repeated small doses of mice Obtaining a high titer anti-human VEGF polyclonal antibody; Step 3: Picking up the mouse, taking the spleen cells, and fusing it with mouse myeloma cells in vitro; Step 4, screening for antibody secretion positive by enzyme-linked immunosorbent assay Hybridoma cells; Step 5. Positive hybridoma cells are subcloned to obtain a plurality of hybridoma monoclonal cells stably secreting anti-human VEGF antibody; Step 6. Expanding and culturing the hybridoma cells, collecting the culture solution, using the affinity layer The anti-human VEGF monoclonal antibody was isolated and purified by an assay.

The preparation method according to claim 3, wherein the step 1 is specifically: PCR-amplifying a gene fragment encoding human VEGF, and cloning it into a yeast expression vector to obtain an expression plasmid, transforming the yeast The high-efficiency expression engineering strain was screened, and the engineered strain was fermented, induced, separated and purified to obtain VEGF protein with a purity of more than 90%.

The method according to claim 3, wherein the step of subcutaneous immunization of the mouse is: mixing the purified recombinant VEGF protein with Freund's complete adjuvant, and subcutaneous injection at a small point. mouse.

The preparation method according to claim 3, wherein the affinity chromatography method of step 6 is specifically: loading the supernatant of the hybridoma cell-containing filtrate containing the monoclonal antibody with the covalent cross-linking After the sample is completed, the affinity chromatography column is eluted with PBS to remove the heteroprotein, and the antibody protein adsorbed by the microparticles is eluted with glycine solution, and then dialyzed and dialyzed. The purified samples were then purified to obtain purified anti-VEGF monoclonal antibodies.

7. An in vitro chemically labeled monoclonal antibody according to claim 2, wherein the labeling step is as follows: 1) dissolving the monoclonal antibody of claim 2 in a PBS solution; 2) adding an effective amount of the label The reagent is mixed with the monoclonal antibody sample, and mixed, and reacted at room temperature; 3), the unreacted excess labeling reagent is removed, and the chemically labeled monoclonal antibody is obtained in vitro.

8. Use of the monoclonal antibody of claim 2 for detecting a VEGF protein.

9. The in vitro chemically labeled monoclonal antibody of claim 7 for detecting VEGF protein use.

10. Use of the monoclonal antibody of claim 2 for isolating VEGF protein.