CN102936283B - Anti-human CD146 monoclonal antibodies, compositions containing anti-human CD146 monoclonal antibodies, and soluble CD146 detection method - Google Patents

Abstract

The invention is a group of anti-human CD146 molecules developed by biotechnology and an established high-sensitivity sandwich ELISA method for detecting soluble CD146. The invention includes: anti-human CD146 mouse monoclonal antibodies AA1, AA2, AA3, AA4, AA5 and AA7, and a method for specifically detecting soluble CD146 by using antibody combination and sandwich ELISA. This group of antibodies can specifically recognize human CD146 at the molecular, cellular and tissue levels, and can be divided into two categories based on their recognition of different epitopes. The sandwich ELISA method combining antibody AA1 with another anti-human CD146 mouse monoclonal antibody AA98 can detect soluble CD146 in the order of gram per milliliter. These antibodies and the detection method will become effective detection or diagnosis tools and methods in basic research or clinical application, and will also provide a good carrier for targeted therapy of CD146-related diseases.

Description

Anti-human CD146 monoclonal antibody, composition comprising same, method for detecting soluble CD146

This application is a divisional application of application number 2008100572607.

technical field

The invention belongs to the fields of molecular biology and biotechnology. Specifically, the present invention relates to a group of mouse monoclonal antibodies against human CD146, which can specifically recognize human CD146 protein at biochemical, cell and tissue levels. The present invention also relates to a method for detecting soluble CD146 by sandwich ELISA. In this detection method, the capture antibody is the mouse monoclonal antibody involved in the present invention, and the detection antibody is the mouse monoclonal antibody involved in the invention patent ZL 991075862 labeled with biotin. Cloned antibody AA98.

Background technique

CD146, also known as MUC18, Mel-CAM/MCAM, is a cell adhesion molecule belonging to the immunoglobulin superfamily. CD146 has five extracellular immunoglobulin-like domains (V-V-C2-C2-C2) (Holness and Simmons1994), which are highly glycosylated and mediate calcium ion-independent intercellular adhesion.

CD146 was first discovered as a marker molecule of melanoma, involved in the metastasis and progression of melanoma (Lehmann, Riethmuller et al.1989; Sers, Kirsch et al.1993). The abnormally high expression of CD146 in melanoma enhances the adhesion between tumor cells and is very important for tumor invasion and metastasis (Johnson, Rothbacher et al.1993). Studies have shown that the expression of CD146 is directly related to the metastatic ability of melanoma cells, and overexpression of CD146 in melanoma cells that do not express CD146 can significantly enhance the invasion and metastatic ability of tumor cells (Bani, Rak et al.1996; Shih, Speicher et al. 1997; Xie, Luca et al. 1997). In addition, CD146 is also expressed in a small amount of normal tissues, such as smooth muscle, vascular endothelium and trophoblast, etc. (Shih1999).

At the same time, CD146 is also widely considered as a specific marker molecule of vascular endothelial cells (Bardin, Frances et al.1996; Bardin, Anfosso et al.2001). In previous studies, anti-human CD146 antibody was used to detect circulating endothelial cells in blood (George, Poncelet et al. 1991; Bardin, George et al. 1996; Solovey, Gui et al. 2001). Because of this feature, CD146 can be used as a reference indicator for disease progression in certain diseases accompanied by endothelial damage and shedding. Other studies have shown that CD146 molecules are also expressed in monocytes, and it has been reported that they are also highly expressed in activated T lymphocytes (Pickl, Majdic et al.1997). Like many other adhesion molecules, the CD146 molecule exists in the cell membrane and in a soluble form (soluble CD146, sCD146) in vivo (Neidhart, Wehrli et al. 1999; Bardin, Moal et al. 2003). Studies have shown that the expression level of sCD146 in synovial fluid of rheumatoid arthritis and serum of patients with chronic renal failure is significantly higher than that of normal people.

Anti-human CD146 antibody has shown a strong therapeutic effect of inhibiting tumor growth in animal experiments. The anti-CD146 fully human antibody developed by Mills et al., ABX-MA1, significantly inhibited the growth, invasion and metastasis of melanoma tumors in a nude mouse model (Mills, Tellez et al.2002). As described in the invention patent ZL 991075862, the developed anti-CD146 mouse monoclonal antibody AA98 has the effect of inhibiting tumor angiogenesis, and significantly inhibited the growth of tumors (such as liver cancer, pancreatic cancer, etc.) in various nude mouse tumor-bearing experiments (Yan , Lin et al. 2003). Further research also revealed that the mechanism of AA98's inhibition of tumor angiogenesis is achieved by inhibiting MAPK phosphorylation and NFκB activation (Bu, Gao et al.2006).

Although some antibodies against human CD146 have been reported, these antibodies have different binding activities to different vascular endothelial tissues. For example, S-endo1 can bind to endothelial cells of various types of blood vessels, including arteries, arterioles, veins, venules, capillaries, high endothelial venules, and lymphatic vasculature (George, Poncelet et al. 1991). Another anti-CD146 antibody, MUC18, only binds to capillaries and high endothelial venules (Kuzu, Bicknell et al. 1993). S-endol binds to umbilical vein endothelium whereas MUC18 does not (Bardin, Frances et al. 1996). In addition, P1H12 can simultaneously bind to vascular endothelial cells of tumor and normal tissues (StCroix, Rago et al.2000), while AA98 specifically binds to vascular endothelial cells of tumor tissues (Yan, Lin et al.2003). These evidences suggest that it is likely that different antibody-binding epitopes are exposed differently in different tissues and microenvironments. Therefore, developing antibodies against different epitopes on CD146 protein and studying their binding ability to different tissues is very necessary to elucidate the function of CD146 on vascular endothelial cells.

Contents of the invention

The invention utilizes natural purified CD146 protein to immunize mice to obtain hybridoma cells. Antibodies with strong binding ability to the extracellular region of the recombinantly expressed CD146 protein were screened by ELISA, and six strains of antibodies were obtained, named AA1, AA2, AA3, AA4, AA5, and AA7, and hybridoma cells secreting these antibodies were obtained at the same time , followed by 8E8, 8F4, A5, G10, H5① and H5②. These six antibodies all belong to IgG1, κ subtype. ELISA and Western blot experiments confirmed the specific binding of these antibodies to CD146 molecules.

Using the different extracellular domains of the recombinantly expressed CD146 protein and immunoblotting, it was confirmed that the AA1 and AA2 recognition epitopes (positions 24-128 in the human CD146 sequence of SEQ ID NO: 1) are located in the first IgV structure domain, while the AA3, AA4, AA5 and AA7 recognition epitopes (positions 335-400 in the human CD146 sequence of SEQ ID NO: 1) are located in the second IgC2 domain. Therefore, these six antibodies are divided into two categories according to different epitopes: V1 category (including AA1 and AA2) and C2-2 category (including AA3, AA4, AA5 and AA7).

Cellular immunofluorescence, immunoprecipitation and immunohistochemical experiments found that V1 antibodies could recognize both native conformation and denatured CD146 protein at the molecular, cellular and tissue levels, while C2-2 antibodies only recognized denatured CD146 proteins. This confirms that different epitopes are exposed differently in different situations.

The present invention uses V1 antibodies, such as AA1 and the biotin-labeled mouse monoclonal antibody AA98 involved in the invention patent ZL991075862, to develop a sandwich ELISA method for detecting soluble CD146 molecules in solutions and body fluids. Compared with the double-antibody sandwich ELISA (sensitivity of 10ng/ml) provided by the commercial detection method report (CyQuant ELISA assay kit, Bioxytex, Marseille, France) provided before, the sensitivity of the sandwich ELISA method of the present invention has been improved by an order of magnitude, reaching 1ng/ml ml. The method can be used to detect soluble CD146 in human blood and cerebrospinal fluid, and has clinical diagnostic application value in diseases with abnormal expression of CD146. Using the method of the present invention, it is found that in autoimmune diseases such as systemic vasculitis, the content of CD146 molecules in the patient's serum is significantly increased, which has guiding significance and application value for clinical diagnosis.

Specifically, the present invention relates to a human CD146 epitope, the amino acid sequence of which is shown in SEQ ID NO: 24 or SEQ ID NO: 25.

In one embodiment, the present invention relates to an anti-human CD146 antibody, which is characterized in that it can specifically recognize the above-mentioned human CD146 epitope.

Preferably, the anti-human CD146 antibody is characterized by comprising an antibody heavy chain and an antibody light chain,

Wherein said antibody heavy chain comprises CDR1 consisting of the amino acid sequence of SEQ ID NO:26 as CDR, CDR2 consisting of the amino acid sequence of SEQ ID NO:27 and CDR3 consisting of the amino acid sequence of SEQ ID NO:28,

The antibody light chain includes, as CDRs, CDR1 consisting of the amino acid sequence of SEQ ID NO:29, CDR2 consisting of the amino acid sequence of SEQ ID NO:30, and CDR3 consisting of the amino acid sequence of SEQ ID NO:31.

More preferably, the heavy chain of the antibody consists of the amino acid sequence of SEQ ID NO:32, and the light chain consists of the amino acid sequence of SEQ ID NO:33.

In another embodiment, the present invention relates to an anti-human CD146 antibody characterized by comprising an antibody heavy chain and an antibody light chain,

Wherein said antibody heavy chain comprises CDR1 consisting of the amino acid sequence of SEQ ID NO:34 as CDR, CDR2 consisting of the amino acid sequence of SEQ ID NO:35 and CDR3 consisting of the amino acid sequence of SEQ ID NO:36,

The antibody light chain includes, as CDRs, CDR1 consisting of the amino acid sequence of SEQ ID NO:37, CDR2 consisting of the amino acid sequence of SEQ ID NO:38, and CDR3 consisting of the amino acid sequence of SEQ ID NO:39.

Preferably, the heavy chain of the antibody consists of the amino acid sequence of SEQ ID NO:40, and the light chain consists of the amino acid sequence of SEQ ID NO:41.

In another aspect of the present invention, it also relates to the application of the above-mentioned anti-human CD146 antibody in the preparation of a pharmaceutical composition for treating tumors. Preferably, said tumor is melanoma, liver cancer, pancreatic cancer.

In another aspect of the present invention, it also relates to the application of the above-mentioned anti-human CD146 antibody in the preparation of a diagnostic agent for imaging localization diagnosis of human tumors targeting CD146.

In another aspect, the present invention also relates to a composition, such as a pharmaceutical composition for treating tumors, which comprises the anti-human CD146 antibody of any aspect above, and a pharmaceutical carrier.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically acceptable. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).

Compositions of the invention can be administered by a number of methods known in the art. As will be appreciated by those skilled in the art, the route and/or mode of administration will vary depending on the desired result.

In order to administer the compounds of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compounds can be administered to a subject in an appropriate carrier, such as liposomes or diluents. Pharmaceutically acceptable diluents include saline and aqueous buffered solutions.

Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.

As used herein, the phrases "parenteral administration" and "parenteral administration" mean modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular , intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, arachnoid Infraspinal, epidural, intrasternal injections and infusions.

Regardless of the chosen route of administration, the compounds of the invention, which may be used in suitable hydrated forms and/or in the form of pharmaceutical compositions of the invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

Actual dosage levels of the active ingredients of the pharmaceutical compositions of this invention will vary for the particular patient, composition and mode of administration to obtain an amount of the active ingredient effective to achieve the desired therapeutic response without being toxic to the patient. The selected dosage level will depend on a number of pharmacokinetic factors, including the activity of the particular composition of the invention used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, and the Other drugs, compounds and/or substances with which a particular compound is used in combination, age, sex, weight, disease condition, general health and previous disease history of the patient to be treated and similar factors well known in the medical arts.

The composition must be sterile and fluid to the extent that the composition can be delivered by syringe. In addition to water, the carrier is preferably an isotonic buffered saline solution.

In another aspect, the present invention also relates to hybridoma cell lines secreting anti-human CD146 antibody, the preservation numbers of which are CGMCC NO: 2310 and CGMCC NO: 2311.

In another aspect, the present invention also relates to a nucleic acid encoding a polypeptide capable of assembling with each of the additional antibody chains defined below, wherein said polypeptide is any of the following polypeptides

a) an antibody heavy chain which is one of the antibody heavy chains defined above;

b) An antibody light chain which is one of the antibody light chains defined above.

At the same time, the present invention also relates to an expression vector comprising a nucleic acid according to claim 11, capable of expressing said nucleic acid in a prokaryotic or eukaryotic host cell. And a prokaryotic or eukaryotic host cell comprising said expression vector.

In another aspect of the present invention, it also relates to a method for preparing an antibody binding to human CD146, which is characterized in that the nucleic acid encoding the heavy chain of the antibody and the nucleic acid encoding the light chain of the antibody are expressed in a prokaryotic or eukaryotic host cell, and from The polypeptide is recovered from the cell.

In another aspect of the present invention, it relates to a method for detecting human CD146, which uses the above-mentioned anti-human CD146 antibody by enzyme-linked immunosorbent assay. Preferably, the enzyme-linked immunosorbent assay is a sandwich enzyme-linked immunosorbent assay, wherein one or more of the above-mentioned anti-human CD146 antibodies are used as capture antibodies, and the mouse monoclonal antibody AA98 in Chinese patent ZL 991075862 is used as a detection antibody Antibody. More preferably, said human CD146 is present in body fluids. Even more preferably, said bodily fluid is interstitial fluid, serum, lymph or cerebrospinal fluid. The body fluid can be collected from patients with autoimmune diseases such as systemic vasculitis, systemic lupus erythematosus, multiple sclerosis and Guillain-Barre syndrome or/and chronic renal failure.

In another aspect of the present invention, it also relates to derivatives of the anti-human CD146 antibody, wherein the derivatives are the antibodies and biomarkers (such as biotin, HRP, alkaline phosphatase, FITC, PE, Cy3, Cy5 and other conventional fluorescent dyes, nano magnetic beads and other nanomaterials), antineoplastic drugs (such as carboplatin, cisplatin, pentafluorouracil, etc. known in the prior art), toxins (such as ricin, lymphotoxin, etc.), Radioactive agents (such as ¹³¹ iodine, ⁹⁰ yttrium, radioactive copper, etc.) combined product.

Those of ordinary skill in the art can obtain the above-mentioned derivatives without difficulty based on the teaching of the anti-human CD146 antibody in the manual and combined with the knowledge of the prior art, and then determine the effect of the derivatives.

The innovations of the present invention are: (1) developed a group of mouse monoclonal antibodies against human CD146 protein; (2) revealed the epitopes recognized by this group of anti-CD146 antibodies, and confirmed the antibodies that recognize different epitopes, There are significant differences in the binding of CD146 protein at the molecular, cellular and tissue levels; (3) A high-sensitivity sandwich ELISA method was developed for the detection of soluble CD146 molecules.

Description of drawings

Figure 1: AA1-AA7 specifically recognizes recombinant human CD146 molecular extracellular segment protein.

Figure 2: Schematic diagram of various CD146 extracellular recombinant proteins.

Figure 3: Epitope identification of AA1-AA5 and AA7. The picture above is the protein electrophoresis of D1-D5. The figure below shows the ability of AA1-AA5 and AA7 to detect their binding ability to D1-D5 by immunoblotting.

Figure 4: Recognition of reduced and non-reduced CD146 protein by AA1-AA5 and AA7 in immunoblots. Lanes 1-6 are non-reduced A375 cell lysates and lanes 7-12 are reduced A375 cell lysates.

Figure 5: AA1-AA5 and AA7 for CD146 molecular detection by flow cytometry. In the figure mIgG was used as a negative control. Only AA1 and AA2 recognize the CD146 molecule in living cells.

Figure 6: AA1-AA5 and AA7 are used in cell immunofluorescence experiments to detect CD146 on the cell membrane. AA98 was used as the positive control of the experiment, and mIgG was used as the negative control of the experiment. Only AA1, AA2 and AA98 can bind to CD146 on the cell membrane, and the arrow shows only one of them.

Figure 7: AA1-AA5 and AA7 were used in immunoprecipitation experiments to capture CD146 in cell lysates. Lanes 1-7 are the protein complexes captured by immunoprecipitation with mIgG, AA1-5 and AA7 respectively. Lane 8 is the supernatant of cell lysate. As a positive control, AA98 is used for immunoblotting detection.

Figure 8: AA1-AA5 and AA7 performed frozen section immunohistochemical experiments to detect CD146 molecules at the tissue level. AA98 was used as the positive control of the experiment, and CD31 was used to label vascular endothelial cells (as indicated by the arrow). As indicated by the arrows, only AA1 and AA2 were able to bind CD146 molecules at the tissue level on cryosections.

Fig. 9: Standard curve for detection of 5ng/ml-320ng/ml standard samples by sandwich ELISA.

Figure 10: Standard curve and fitting equation for detection of 5ng/ml-80ng/ml standard samples by sandwich ELISA.

Figure 11: Map of the HMV6g vector, which contains the MBP-His tag.

Hybridoma cell lines 8E8 and G10 (which secrete antibodies AA1 and AA4, respectively) were deposited in the General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC, Datun Road, Chaoyang District, Beijing, Microbiology Research, Chinese Academy of Sciences) on December 28, 2007. Institute 100101), classified as mouse monoclonal antibody hybridoma cells, and the preservation numbers are CGMCC No.2310 and CGMCC No.2311.

Detailed ways

The present invention is described in detail below by way of examples. Those of ordinary skill in the art will appreciate that the following examples are for illustration purposes only. The spirit and scope of the invention are defined by the appended claims.

Example 1: Preparation and identification of monoclonal antibodies AA1-AA5 and AA7.

Application of hybridoma technology (Kohler and Milstein1975; Yeh, Hellstrom et al.1979; Yeh, Hellstrom et al.1982) produced and screened to obtain antibodies AA1-AA6 and AA7. The brief description is as follows: Isolate natural CD146 protein (its amino acid sequence is shown in SEQ ID NO: 1, and its nucleotide sequence is shown in SEQ ID NO: 2) from human umbilical cord vein endothelial cells, according to (Yan, Lin et al.2003 ) to purify the monoclonal antibody AA98 antigen, and use it as an immunogen to immunize BALB/C mice (Beijing Experimental Animal Center), inject 100 μg protein/mouse intraperitoneally, once every two weeks, for a total of three times. Before the splenocytes were collected, immunization was boosted once, and 100 μg protein/mouse was injected intraperitoneally. Three days after the booster immunization, the spleen was taken, and the splenocytes were suspended in RPMI medium. Splenocytes were fused with SP2/0-Ag14 murine myeloma cells (ATCC) in the presence of polyethylene glycol (PEG), and hybridomas were selected using HAT-selective medium.

The method of enzyme-linked immunosorbent assay (ELISA) was used to screen hybridoma cell clones capable of producing a large number of high-affinity anti-human CD146 antibodies. The recombinantly expressed extracellular domain protein of CD146 molecule, rhCD146 (position amino acid 24-552 in the human CD146 sequence of SEQ ID NO: 1) was used as a coating antigen to detect hybridoma cell culture supernatant. The specific method is first to coat 50 μl of 1 μg/ml rhCD146 protein on the ELISA plate overnight, and wash it three times with PBS. 2% bovine serum albumin (Ameresco) was added to block for 1 hour. Then, the hybridoma culture supernatant secreting the antibody, enzyme-labeled antibody (1:5000 diluted goat anti-mouse HRP, Santa Cruz) and substrate (200ng/ml TMB (Ameresco), 0.03% H ₂ O ₂ (Beijing Chemical Reagent Company), pH 4.5), for ELISA screening. Six hybridoma cell lines 8E8, 8F4, A5, G10, H5① and H5② were obtained through three rounds of screening, and the secreted antibodies were named AA1, AA2, AA3, AA4, AA5 and AA7 respectively. The hybridoma cell lines 8E8 and G10 (which secrete antibodies AA1 and AA4 respectively) were deposited in the General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC, Datun Road, Chaoyang District, Beijing, Microbiology, Chinese Academy of Sciences) on December 28, 2007. Research Institute 100101), classified as mouse monoclonal antibody hybridoma cells, and the preservation numbers are CGMCC No.2310 and CGMCC No.2311. The hybridoma cell line AA6 is a negative hybridoma clone in ELISA screening, and its ability to bind to CD146 is very weak.

The hybridoma cells 8E8, 8F4, A5, G10, H5①, and H5② were cultured and expanded in large quantities, and the culture supernatants containing AA1, AA2, AA3, AA4, AA5, and AA7 antibodies were collected for antibody function identification. In addition, the hybridoma cells 8E8, 8F4, A5, G10, H5① and H5② were respectively made into cell suspensions in serum-free RPMI-1640 medium for the preparation of antibody ascites. The preparation method of ascites is briefly described as follows: six-week-old BALB/C mice were intraperitoneally injected with 0.5 ml pristane (Sigma) per mouse. Ten days later, inoculate the hybridoma cell suspension at 1×106 cells/ml in the peritoneal cavity of BALB/C mice at 0.5 ml/mouse. After about ten days, collect the ascites and centrifuge to get the supernatant.

Monoclonal antibodies AA1, AA2, AA3, AA4, AA5 and AA7 were purified from culture supernatant or ascites fluid by protein A affinity chromatography. Purified monoclonal antibodies are sterile filtered and stored refrigerated or frozen.

Using BD Pharmingen's mouse antibody subtype identification kit (catalogue number 550487), using AA98 (the same below) described in the invention patent ZL 991075862 as the positive control of the experiment, antibodies AA1, AA2, AA3, AA4, AA5 and All AA7 belong to the IgG1 subtype, as shown in Table 1.

Table 1 Types of antibodies AA1, AA2, AA3, AA4, AA5, AA7 and AA98

Antibody heavy chain subtype light chain subtype AA1 IgG1 kappa AA2 IgG1 kappa AA3 IgG1 kappa AA4 IgG1 kappa AA5 IgG1 kappa AA7 IgG1 kappa AA98 IgG2a kappa

The ELISA method was used to identify the binding specificity of the purified AA1-AA5 and AA7 antibodies to CD146 protein. The His-rhCD146 of the 6xHis-tag fusion of prokaryotic expression (by 6xHis-tag and sequence is the amino acid of position 24-552 in the human CD146 sequence of SEQ ID NO: 1 is made up of) and the alkaline phosphatase (Alkaline Phosphatase) of eukaryotic expression , referred to as AP, its sequence is shown in SEQ ID NO: 3) fused sCD146-AP (the amino acid at position 24-559 in the human CD146 sequence whose sequence is SEQ ID NO: 1 and the sequence is SEQ ID NO: 3 AP and the linker region whose sequence is SEQ ID NO: 42) were respectively used as coating antigens in the experiment (see Figure 2 for their structural schematic diagrams).

Among them, the preparation method of His-rhCD146 is briefly introduced as follows: the nucleotides at positions 70-1656 in the cDNA sequence (SEQ ID NO: 2) of human CD146 are cloned onto the pET28b (Novagen) vector, and the restriction enzymes NdeI and NotI (provided by NEB) and primers 5'-AA C ATA TG G TGC CCG GAG AGG CTG AGC AG-3'(SEQ ID NO:4) and 5'-AAGCGG CCG CCA GCT TTC TCT CTG TGG AG-3'(SEQ ID NO:5). It is expressed in E.ColiBL21 DE3 (Invitrogen) strain and exists in the form of inclusion bodies, and the desired antigen can be obtained after washing and renaturation. For protein expression and inclusion body washing methods, see D1, D2 and D4 below for protein expression and inclusion body washing methods.

The steps for renaturation of inclusion bodies are as follows:

1. Adjust the concentration of the dissolved inclusion body protein to 1mg/ml, put 1ml in total into the dialysis bag, and use 140ml of 6M urea, 200mM arginine, 25mM Tris (pH8.0), 150mM NaCl, 2mM reduced gluten as the external solution Glutathione (GSH), 1mM oxidized glutathione (GSSG) were dialyzed overnight at 4°C.

2. Pour out the above 50ml of dialyzed fluid, and add 50ml of diluent [600mM arginine, 25mM Tris (pH8.0), 150mM NaCl, 2mM GSH, 1mM GSSG.]. At this time, the urea concentration of the external fluid is 4M. Dialyze at 4°C for 6 hours.

3. Pour out 75ml of dialysis fluid, add 75ml of diluent, and the final concentration of urea is 2M. Dialyze at 4°C for 6 hours.

4. With 200ml 400mM arginine, 25mM Tris, 150mM NaCl, 2mM GSH, 1mM GSSG, dialyze overnight. The concentration of urea was 0M and that of arginine was 400mM.

5. Pour out 100ml of dialysis fluid, add 100ml of 25mM Tris, 150mM NaCl. The final solution was 200mM Arginine, 25mM Tris, 150mM NaCl, 1mM GSH, 0.5mM GSSG. Dialyze at 4°C for 6 hours.

6. Repeat step 5.

7. Dialyze with 900ml25mM Tris150mM NaCl overnight.

8. Change to fresh 1L 25mM Tris150mM NaCl and dialyze for 6 hours.

9. Determination of protein content, detection by SDS-PAGE method. The concentration can reach more than 500μg/ml. It can be further concentrated by ultrafiltration or Ni-NTA affinity chromatography. The chemical reagents used for dialysis were provided by Sigma. )

The preparation process of sCD146-AP is simple as follows: the coding nucleotide sequence (SEQID NO: 6) of the fusion protein is cloned on the pCMV-SPORTS6 vector (provided by OpenBiosystems) with XbaI and EcoRI (NEB), and the plasmid containing the insert fragment is cloned using Fugene6 ( Roche) transfection reagent was transiently transfected into 293T cells (ATCC), and the cell culture supernatant was purified according to the monoclonal antibody AA98 antigen purification method described in (Yan, Lin et al. 2003) to obtain sCD146-AP fusion protein.

Use ELISA to detect the combination of antibody and recombinant protein, the simple method is as follows.

1) Preparation of Trx-His (its amino acid sequence is shown in SEQ ID NO: 7): According to the method for expressing D3 and D5 proteins in Example 2, pET32a (Novagen) is transformed into B121 (DE3) competent cells, using IPTG Induced expression, purified by Ni column. Thrombin protease (15 U enzyme/mg protein, provided by Sigma) was used for enzymatic digestion at 4°C overnight, and then the Trx-His with 6xHis tag was purified by Ni-NTA, and the method was similar to that of purifying D3 and D5 proteins in Example 2.

2) Preparation of MBP-His (its amino acid sequence is shown in SEQ ID NO: 8): According to the method for expressing D3 and D5 proteins in Example 2, the HMV6g vector (vector map as shown in Figure 11) is transformed into Bl21 (DE3 ) Competent cells were induced by IPTG and purified by Ni column. Tev protease (1 μg enzyme/mg protein, provided by Invitrogen) was used for enzymatic digestion at 4°C overnight, and then the MBP-His with 6xHis tag was purified by Ni-NTA, and the method was similar to that of Example 2 for purifying D3 and D5 proteins.

3) Coat 1 μg/ml antigen (His-rhCD146, sCD146-AP, Trx-His and MBP-His) on different wells of the ELISA plate, 50 μl/well, overnight at 4°C; use 2% BSA/PBS (Ameresco) was blocked at room temperature for 2 hours, and then incubated with 50 μl/well of 1:2000 diluted antibody (prepared ascites AA1-5 and AA7) on the plate for 2 hours at room temperature, followed by washing 5 times with PBST and 1 time with PBS. The secondary antibody was incubated with 1:5000 diluted HRP-conjugated goat anti-mouse IgG (Santa Cruz) for 1 hour at room temperature, washed 5 times with PBST and 1 time with PBS. TMB substrate (200 ng/ml TMB (Ameresco), 0.03% H ₂ O ₂ (Beijing Chemical Reagent Company), pH 4.5), 100 μl/well was used for color development, and sulfuric acid was used to stop. The results are shown in Figure 1. Both AA1-AA5 and AA7 can strongly bind to the extracellular region of recombinantly expressed CD146 protein: His-rhCD146 and sCD146-AP, but do not bind to 6xHis-tag fused thioredoxin (Trx -His) and 6xHis-tag fused mannose-binding protein (MBP-His). This confirmed that all six antibodies specifically recognized CD146 protein rather than His-tag. AA6 was used as the negative control of the experiment.

Example 2: Epitope identification of monoclonal antibodies AA1-AA5 and AA7.

The antigenic epitopes of the six antibodies described in the present invention were identified by recombinantly expressing the five domain proteins of the extracellular region of human CD146 and immunoblotting.

As shown in Figure 2, domains 1-5 respectively represent the five domains V-V-C2-C2-C2 of CD146 from the extracellular to the transmembrane region. Overlapping sequences were designed to prevent possible loss of epitopes. Structural domain 1 (the sequence is the amino acid of position 24-145 in the human CD146 sequence of SEQ ID NO: 1, as shown in SEQ ID NO: 9) is cloned on pET30a (Novagen), what expresses is His6-Stag fusion His6 - S-D1 protein. Structural domain 2 (sequence is the amino acid of position 128-248 in the human CD146 sequence of SEQ ID NO:1, as shown in SEQ ID NO:10), structural domain 3 (sequence is the human CD146 sequence of SEQ ID NO:1 The amino acid of the position 233-335 of the position, as shown in SEQ ID NO:11), structural domain 4 (sequence is the amino acid of position 313-442 in the human CD146 sequence of SEQ ID NO:1, as shown in SEQ ID NO:12) And domain 5 (sequence is the amino acid of position 400-560 in the human CD146 sequence of SEQ ID NO: 1, as shown in SEQ ID NO: 13) clones on pET32a (Novagen) respectively, what expresses is respectively Trx-His6 -S tag fused Trx-His6-S-D2, Trx-His6-S-D3, Trx-His6-S-D4 and Trx-His6-S-D5.

The cloning process is briefly described as follows: use the corresponding pair of primers D1S and D1A (for domain 1), D2S and D2A (for domain 2), D3S and D3A (for domain 3), D4S and D4A (for domain 3), and for structural domain 4), D5S and D5A (for structural domain 5), and template pcDNA3.1(-)b-CD146 (using CD146cDNA derived from Johnson, J.P. laboratory with sequence as shown in SEQ ID NO: 2 Restriction endonucleases EcoR I and BamH I (NEB) were cloned into pcDNA3.1(-)b vector (Invitrogen) for PCR reaction, the conditions were 95°C for 5 minutes; 95°C for 45 seconds, 56°C for 40 seconds, 72°C for 50 seconds, 30 cycles; 72°C for 5 minutes. Nucleotide sequences encoding domains 1-5 were amplified, respectively.

The list of primers is as follows, the sense strand primer has a Nco I restriction site (as shown in the underline), and the antisense strand primer has a Hind III restriction site (as shown in the underline):

The PCR product was separated with 2% agarose gel (Biowest Agarose), and the PCR product was recovered with the agarose gel DNA recovery kit (product number: DP209-03) provided by Tiangen Company, and then the pMD18T-simple kit provided by Takara Company was used to The fragment was ligated to T vector (vector and enzyme provided in the kit), and Top10 competent cells (provided by Tiangen) were transformed. The monoclonal D1-T (the nucleotide sequence inserted into the structural domain 1), D2-T (the nucleotide sequence inserted into the structural domain 2), D3-T ( The nucleotide sequence of domain 3 was inserted), D4-T (the nucleotide sequence of domain 4 was inserted) and D5-T (the nucleotide sequence of domain 5 was inserted), with ampicillin The LB medium was cultured at 37°C overnight, and the plasmid was extracted with the plasmid mini-extraction kit (product number: DP103-03) provided by Tiangen, and pET30a, pET32a, and extracted plasmids (i.e. T vector into which the nucleotide sequences of domains 1-5 were inserted, respectively). Afterwards, use 2% agarose gel (Biowest Agarose) to separate, and use the agarose gel DNA recovery kit (product number: DP209-03) provided by Tiangen Company to reclaim the enzyme-cut small fragment of the T vector (i.e. domains 1-5 nucleotide sequence) and pET30a and pET32a enzyme cut plasmid fragments. The small enzyme-cut fragments of D1-T were ligated with the plasmid fragments cut by pET30a enzyme, and the small enzyme-cut fragments of D2-T, D3-T, D4-T and D5-T were connected with the plasmid fragments cut by pET32a enzyme, and used NEB The T4 DNA ligase provided by the company was used to establish the ligation reaction, and ligated overnight at 16°C. Transform Top10 competent cells (Tiangen), pET30a-D1 was screened with semi-solid LB plates containing kanamycin (50ng/ml), pET32a-D2, pET32a-D3, pET32a-D4, and pET32a-D5 were screened with ampicillin Mycin (100ng/ml) semi-solid LB plate selection. The single clones obtained by overnight culture were cultured in LB medium containing antibiotics, and the plasmids were extracted, and the obtained plasmids were used to transform Bl21(DE3) competent cells (Tiangen).

Induced expression of recombinant proteins His6-S-D1, Trx-His6-S-D2, Trx-His6-S-D3, Trx-His6-S-D4 and Trx-His6-S-D5: transformed pET30a-D1, pET32a -D2, pET32a-D3, pET32a-D4, and pET32a-D5 Bl21(DE3) single clones were cultured overnight at 37°C in 5ml LB medium containing the corresponding antibiotics. Overnight bacteria were inoculated into the corresponding fresh medium at 1:100 and cultured at 37°C. When the bacteria grew to an OD600 of 0.6, 1mM IPTG (Ameresco) was added to induce expression at 37°C for 6 hours or 0.2mM IPTG was induced overnight at 16°C.

After the expression of D1, D2 and D4 was induced by IPTG, they were in the state of inclusion body and insoluble. Inclusion body purification and washing methods are as follows: bacteria resuspended in 25mM Tris-HCl (pH7.4), 300W ultrasonic 2x99 cycles, each cycle ultrasonic 4 seconds, pause 8 seconds, a total of 12 seconds. Centrifuge at 12000 g for 30 minutes at 4°C. The inclusion body pellet was resuspended in Solution 1 (2.5M NaCl), stirred at 4°C for 30 minutes, and centrifuged at 12,000 g for 30 minutes at 4°C. The inclusion body pellet was resuspended in Solution 2 (0.5% Triton X-100, 10mM EDTA, pH 8.0), stirred at 4°C for 30 minutes, and centrifuged at 12000g at 4°C for 30 minutes. The inclusion body pellet was resuspended in Solution 3 (2M Urea, 50mM Tris, 1mM EDTA, pH8.0), stirred at 4°C for 30 minutes, and centrifuged at 12000g for 30 minutes at 4°C. Subsequently, inclusion bodies were dissolved in solution 4 (8M Urea, 25mM Tris, 150mM NaCl, 25mM DTT, pH 8.0).

After the expression of D3 and D5 was induced by IPTG, they were in soluble state and purified by Ni column. The specific method is as follows: bacteria resuspended in 25mM Tris-HCl (pH7.4), 300W ultrasonic 2x99 cycles, each cycle ultrasonic 4 seconds, pause 8 seconds, a total of 12 seconds. Centrifuge at 12,000 g at 4°C for 30 minutes, collect the supernatant, and pass through a 0.45 mm filter to remove impurities. Ni-NTA sepharose6 Fast Flow (GE HealthCare) was used to fill the column, washed with ultrapure water, and then equilibrated in an equilibrium solution of 50mM Tris-HCl (pH7.4), 150mM NaCl. Then the centrifuged soluble supernatant containing His6-tag protein was loaded onto the column, and the column was washed with washing solution (50mM Tris-HCl, pH7.4, 150mM NaCl, 50mM imidazole). The final elution solution (50mM Tris-HCl, pH7.4, 150mM NaCl, 300mM imidazole) elutes the protein on the column.

The antigenic epitopes recognized by AA1-AA5 and AA7 were identified by immunoblotting. The simple process is as follows: use 15% SDS-PAGE to separate the prokaryotic expressed and purified D1-D5 proteins, and then use the Bio-rad semi-dry transfer instrument to transfer the proteins on the gel to the nitrocellulose membrane. Block with 5% skimmed milk dissolved in PBST at room temperature for 2 hours, and then incubate overnight at 4°C with the primary antibody solution (AA1-AA5 and AA7) diluted in 5% skimmed milk/PBST (1:2000) above. Then wash 5 times with PBST, and incubate at room temperature for 1 hour with the secondary antibody (horseradish peroxidase-coupled goat anti-mouse IgG antibody, Pierce) diluted in 5% skimmed milk/PBST (1:2000). After washing with PBST for 5 times, the chromogenic substrate of Pierce Company (product number: 34076, 400 μl 3% H ₂ O ₂ +400 μl Luminol solution/miniblot) was used for color development. In the experiment, AA1, AA2, AA3, AA4, AA5, and AA7 were used as the primary antibody, and horseradish peroxidase-coupled goat anti-mouse IgG (Pierce) was used as the secondary antibody to detect the presence of recombinantly expressed CD146 extracellular Binding activity of the five domains D1-5. As shown in Figure 3, AA1 and AA2 only bind to D1 and do not bind to the adjacent D2, confirming that their antigenic epitopes are between positions 24-128 in the human CD146 sequence whose sequence is SEQ ID NO:1, the specific sequence is as follows Shown in SEQ ID NO:24. AA3-5 and AA7 only bind to D4, and do not bind to adjacent D3 and D5, confirming that their antigenic epitopes are between positions 335-400 in the human CD146 sequence whose sequence is SEQ ID NO: 1, and the specific sequence is as shown in SEQ ID Shown in NO:25.

These six antibodies were divided into two categories based on different epitopes, V1 and C2-2. Because the epitopes of AA1 and AA2 are located in the first IgV-like domain, they are classified as class V1, while the epitopes of AA3, AA4, AA5, and AA7 are located in the second IgC2-like domain, so they are classified as class C2-2 .

Example 3: Detection of human CD146 using monoclonal antibodies AA1-AA5 and AA7

The anti-human CD146 mouse monoclonal antibody of the present invention can detect human CD146 protein at molecular, cell and tissue levels.

In whole-cell western blot experiments, both AA1-AA5 and AA7 could recognize CD146 protein in both reduced and non-reduced states. The specific experimental method is as follows: collect human melanoma cells A375 (ATCC) that highly express CD146, wash the cells twice with pre-cooled PBS, centrifuge at 800 rpm at 4°C for 5 minutes, and use lysate (Tris-HCl 50mM pH8.0 , NaCl150mM, EDTA1mM, NP-401%, Glycerol10%, PMSF100μg/ml) to lyse the cells, centrifuge at 12000g at 4°C for 15 minutes, collect the supernatant, add DTT (final concentration 100mM) (dithiothreitol) and no DTT sample buffer (5x sample buffer: 0.313M Tris-HCl, pH 6.8, 10% SDS, 0.05% bromophenol blue, 50% glycerol), cook at 100°C. Whole-cell proteins were separated by 10% SDS-PAGE, followed by semi-dry electrotransfer to nitrocellulose membranes. After blocking with 5% skim milk, use AA1-AA5 and AA7 as primary antibodies (1:10000 diluted with blocking solution 5% skim milk/TBST) to incubate the membrane overnight at 4°C, and then use horseradish peroxidase-coupled sheep Anti-mouse IgG secondary antibody (Pierce, 1:2000 blocking solution 5% skimmed milk/TBST dilution) was incubated at room temperature for 1 hour, and the chromogenic substrate provided by Pierce (product number: 34076, 400 μl 3% H ₂ O ₂ +400 μl Luminol solution/miniblot) detection. As shown in Figure 4, AA1-AA5 and AA7 can bind to the CD146 protein molecule expressed by A375 at the molecular level.

Using flow cytometry detection methods, it was confirmed that V1 antibodies could recognize the CD146 protein on the cell membrane surface at the living cell level, while C2-2 antibodies could not. The specific experimental method is as follows: human melanoma cells A375 (same as above) highly expressing CD146 were collected, the cells were washed twice with pre-cooled PBS containing 0.3% BSA, and centrifuged at 800 rpm at 4°C for 5 minutes after each wash. The cells were blocked with goat serum dilution (1:20 diluted in PBS), then AA1-AA5 and AA7, and the positive control AA98 were added as primary antibodies (1:2000 diluted in PBS), and incubated on ice for 40 minutes. Wash the cells twice with pre-cooled PBS containing 0.3% BSA, add FITC-coupled goat anti-mouse IgG as the secondary antibody (Sigma, 1:300 dilution in PBS), incubate at room temperature for 20 minutes, and detect with the BD FACSCalibur flow cytometry system (BD Bioscience). The results are shown in Figure 5. AA1 and AA2, namely V1 antibodies, can bind to the CD146 molecule expressed by A375, which is significantly different from the negative control mIgG (mouse IgG was used as the primary antibody, provided by Sigma). However, AA3-5 and AA7, that is, C2-2 antibodies, cannot bind to the natural state CD146 molecules on the A375 membrane of living cells. The possible reason is that the antigenic epitopes recognized by C2-2 are not exposed to CD146 proteins in the natural state. the surface of the molecule.

Similarly, using immunofluorescence technology experiments, only V1 antibodies can recognize CD146 molecules at the cellular level, while C2-2 antibodies cannot. AA98 is used as the positive control of the experiment, and mIgG is used as the negative control, as shown in Figure 6. Experiments also confirmed that CD146 molecules are mainly distributed on the cell membrane surface. The experimental procedure is briefly described as follows: A375 cells longer than 24-well plates were fixed with pre-cooled 1:1 acetone/methanol for 1 minute, and then washed twice with PBS. The cells were blocked with 2% goat serum (Beijing Zhongshan Jinqiao) at 37°C for 30 minutes, and washed twice with PBS. Then add 100 μl of primary antibodies (AA1-AA5 and AA7, AA98 and mIgG) diluted 1:2000 in PBS, and incubate at 37°C for 2 hours. After washing with PBS, FITC-conjugated goat anti-mouse IgG (Sigma) diluted 1:300 in PBS was used as the secondary antibody and the cells were incubated at 37° C. for 1 hour. Wash with PBS and examine under microscope.

Antibodies of class V1 can be used to enrich native CD146 molecules in solution. Using the immunoprecipitation technique, the V1 antibody can bind and enrich the CD146 protein in the cell lysate. The specific method is as follows: 1×107A375 (same as above) cells were lysed in 0.6 mL ice-cold above-mentioned lysate for 30 minutes, centrifuged at 12000 g at 4° C. for 15 minutes, and the supernatant was collected. Add 20 μl of 50% slurry (50% mixed with PBS or TE) protein A-Agarose (Santa Cruz) for pre-clearing. Then add 2 μg AA1-AA5 and AA7 respectively, and incubate overnight at 4°C. Then add 20 μl 50% slurry of protein A-Agarose to capture the antigen-antibody complex, wash the precipitate three times with PBS, add 40 μl 1x loading buffer (dilute the above 5x loading buffer 5 times), heat and cook the sample at 100°C for 10 minutes. Protein precipitation was detected by immunoblotting (the experimental method was the same as above), and AA98 was used as the primary antibody for detection. As shown in Figure 7, AA1 and AA2 were able to precipitate and enrich CD146 protein in A375 lysate, while C2-2 class antibodies could not.

Using immunohistochemical techniques, V1 antibodies can detect CD146 protein molecules at the tissue level. The specific method is as follows: the human umbilical cord tissue was embedded with OCT (frozen tissue embedding solution, provided by Sakura), and frozen sections were performed. Sections were fixed in pre-cooled acetone for 5 minutes, and then incubated in methanol containing 0.3% H ₂ O ₂ for 30 minutes at room temperature in the dark to remove the interference of endogenous peroxidase. After washing with PBS three times, the sections were blocked with 5% horse serum (Beijing Zhongshan Jinqiao, horse serum diluted in PBS), and then the sections were treated with primary antibody dilutions containing AA1-AA5, AA7, AA98 and anti-CD31 antibody (Santa Cruz) (1: 2000 dilution in blocking solution) and incubated overnight at 4°C. Then use biotin-labeled secondary antibody (biotin-coupled goat anti-mouse IgG, Vector company, 1:1000 dilution in blocking solution) and horseradish peroxidase-coupled streptavidin as the third antibody (Vector , 1:1000 PBS dilution), incubate tissue sections at 37°C, and finally develop color with a freshly prepared DAB color development kit (Beijing Zhongshan Jinqiao, product number: ZLI-9033). After color development, nuclei were counterstained with hematoxylin. As shown in Figure 8, anti-CD31 antibody labeled vascular endothelial cells (as indicated by the arrow), AA1 and AA2 could recognize CD146 molecules distributed in umbilical cord vein endothelial cells and vascular smooth muscle cells at the tissue level (as indicated by the arrow), while C2 -2 class antibodies cannot. AA98 was used as the positive control of the experiment (as indicated by the arrow).

Example 4: High-sensitivity double-antibody sandwich ELISA method for detecting human soluble CD146

Using the anti-human CD146 antibody AA1 described in the present invention and the AA98 described in the invention patent ZL 991075862, the present invention has developed a highly sensitive double-antibody sandwich ELISA method to detect soluble CD146 in serum. The main method is to use AA1 (the experiment on AA2 is the same, so it is omitted) as the capture antigen, biotin-labeled AA98 as the detection antigen, and eukaryotic expressed sCD146-AP as the standard to detect normal people and patients with autoimmune diseases Soluble CD146 protein in serum. Experiments have confirmed that the sandwich ELISA developed by the present invention has higher sensitivity and wider linear range. Using this method, it has also been confirmed that the CD146 protein content in the serum of patients with autoimmune diseases such as systemic vasculitis is significantly higher than that of healthy volunteers.

The specific experimental method is as follows:

1. Sample source and clinical data: The plasma of healthy volunteers came from the Beijing Red Cross Blood Center, and the plasma of patients with vasculitis came from Anzhen Hospital.

2. Specific steps:

1) Antibody coating on ELISA plate: Dilute antibody AA1 to 1 μg/ml with 0.02M PB (pH7.25), coat 50 μl/well overnight at 4°C.

2) Block non-specific binding sites: 200 μl/well 2% BSA/PBS, incubate at room temperature for 2 hours.

3) Sample incubation: Dilute the known concentration of sCD146-AP gradient with blocking solution (2%BSA/PBS) to draw a standard curve, and the diluted concentrations are 5, 10, 20, 40, 80, 160, 320ng /ml. The serum to be tested was diluted 20 times, and the loading volume was 50 μl, and the blocking solution replaced the sample as a negative control. Each sample was replicated twice. Incubate at room temperature for 2 hours.

4) PBST 5 times, PBS 1 time to wash away non-specific binding.

5) Detection antibody incubation: detection antibody AA98-biotin (biotin used to label AA98 provided by Pierce) was diluted to 0.5 μg/ml with 2% BSA/PB, and 50 μl/well was incubated at room temperature for 2 hours.

6) PBST 5 times, PBS 1 time to wash away non-specific binding.

7) Enzyme-labeled secondary antibody amplification signal: add streptavidin-horseradish peroxidase (Vector, 1:3000) at an appropriate concentration, 50 μl/well, and incubate at room temperature for 1 hour.

8) PBST 5 times, PBS 1 time to wash away non-specific binding.

9) Color reaction: add substrate (200ng/ml TMB (Ameresco), 0.03% H ₂ O ₂ (Beijing Chemical Reagent Company), pH 4.5) for color development, 100 μl/well, 37°C for 15 minutes, add 50 μl/well 2M H ₂ SO ₄ terminated the reaction, and read on a microplate reader at 450nm.

10) Software statistical results: the software analyzes the absorbance value, and draws a standard curve according to the standard sCD146-AP concentration.

11) Calculate the concentration of the sample to be tested according to the standard curve.

3. Result analysis:

1) Make a standard curve using the known concentration of sCD146-AP. The concentration of sCD146-AP after dilution was 5-320 ng/ml, and the standard curve was drawn as shown in FIG. 9 . It was calculated that the detection range of the method was linear within 5-80 ng/ml. The standard curve (R ² =0.9981) obtained by plotting the sCD146-AP concentration of 5-80 ng/ml is shown in FIG. 10 .

2) Analyze the plasma sCD146 content of healthy volunteers and patients with systemic vasculitis. The plasma sCD146 content of healthy volunteers was 154.7 ng/ml, while the plasma sCD146 content of patients with vasculitis reached 383.9 ng/ml. The difference between the two is obvious, as shown in Table 2.

Table 2: Contents of soluble CD146 in serum of healthy volunteers and patients with systemic vasculitis detected by sandwich ELISA.

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Claims (15)

1. Aminoacid sequence shown in the 313-442 position of 1.SEQ ID NO:1.
2. 2. an anti-human CD146 antibody, is characterized in that the aminoacid sequence that it can specific recognition claim 1, and its hybridoma cell strain that is CGMCC NO.2311 by preserving number is secreted.
3. 3. the application of the antibody of the anti-human CD146 of claim 2 in the pharmaceutical composition for the preparation for the treatment of tumour, wherein said tumour is melanoma, liver cancer, carcinoma of the pancreas.
4. 4. the application of the antibody of the anti-human CD146 of claim 2 in the diagnostic reagent of the imaging level diagnosis of the human tumor for the preparation of target CD146.
5. 5. a pharmaceutical composition that is used for the treatment of tumour, anti-human CD146 antibody and pharmaceutical carrier that it comprises claim 2.
6. 6. secrete the hybridoma cell strain of anti-human CD146 antibody, preserving number is CGMCC NO:2311.
7. 7. the nucleic acid of anti-human CD146 antibody described in the claim 2 of encoding.
8. 8. comprise according to an expression vector for the nucleic acid of claim 7, it can express described nucleic acid in protokaryon or eukaryotic host cell.
9. 9. protokaryon or an eukaryotic host cell, it comprises the carrier according to claim 8.
10. 10. prepare the method in conjunction with the antibody of people CD146, it is characterized in that expressing according to the nucleic acid of the nucleic acid of the encoding antibody heavy chain of claim 7 and encoding antibody light chain in protokaryon or eukaryotic host cell, and from described cell, reclaim described polypeptide.
11. The application of the anti-human CD146 antibody of 11. claims 2 in the diagnostic reagent for the preparation of detection people CD146, wherein said detection is undertaken by enzyme-linked immunosorbent assay.
12. The application of 12. claims 11, wherein said enzyme-linked immunosorbent assay is sandwich enzyme-linked immunoabsorption, wherein using the anti-human CD146 antibody of claim 2 as capture antibody, using the mouse monoclonal antibody AA98 in Chinese patent ZL991075862 as detecting antibody.
13. 13. claims 11 or 12 application, wherein said people CD146 is present in human body fluid.
14. The application of 14. claims 12, wherein said body fluid is tissue juice, serum, lymph liquid or cerebrospinal fluid.
15. The derivative of the antibody of the anti-human CD146 of 15. claims 2, wherein said derivative is the product that described antibody is combined with biomarker, antitumor drug, toxin, radioactivity agent, wherein said biomarker is vitamin H, horseradish peroxidase, alkaline phosphatase, fluorescein isothiocyanate, phycoerythrobilin, Cy3, Cy5 or nanometer magnetic bead, described antitumor drug is carboplatin, cis-platinum or 5-Fluorouracil, toxin is Ricin or lymphotoxin, and radioactivity agent is ¹³¹iodine, ⁹⁰yttrium or radioactivity copper.