Anti-b7h3 protein monoclonal antibody and preparation method and application thereof

Abstract

The present application relates to a kind of monoclonal antibody that can identify human B7H3 antigen, its preparation method and its use in immunodetection.The present application provides a kind of anti-B7H3 protein rabbit monoclonal antibody, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1;The amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.2.The antibody has high specificity, sensitivity, and can specifically recognize the cell expressing B7H3 protein, is suitable for immunological detection, especially immunohistochemical detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical engineering, and particularly relates to an anti-B7H3 protein monoclonal antibody and a preparation method and application thereof. BACKGROUND

[0002] B7H3 is a member of the B7 family of costimulatory molecules, one of the immune checkpoint molecules of the B7-CD28 family. It plays an important role in T cell-mediated immune response. B7H3 protein is not expressed or very low expressed in normal tissues and cells, but is abnormally expressed in various malignant tumors, and is closely related to the progression of tumors and the prognosis of patients. B7H3 protein molecule is a type I transmembrane protein encoding 316 amino acids, containing 7 exons and 6 introns. In addition, there are two main splice bodies of B7H3 protein, B7H3a (2Ig-B7H3) with extracellular segment composed of Ig-V-IgC and B7-H3b (4Ig-B7H3) with extracellular segment composed of Ig-V-IgC-Ig-V-IgC, and humans mainly express 4Ig-B7H3.

[0003] The role of B7H3 in tumor immunity is controversial. On the one hand, it can play a costimulatory role, stimulate the proliferation of CD4 and CD8 T cells and the secretion of cytokines such as IFN-γ, IL-8 and TNF-α, and improve the immune response of the body to tumors. On the other hand, it can inhibit the activation of CD4+ T cells and the production of cytokines such as IFN-γ and IL-4, and play a costimulatory role.

[0004] Studies have confirmed that the expression level of B7H3 is closely related to the occurrence, proliferation, metastasis and invasion, metabolism and tumor angiogenesis of malignant tumors. Immunohistochemical results have shown that B7-H3 is highly expressed in liver cancer, colorectal cancer, endometrial cancer, breast cancer, bile duct cancer, colorectal cancer and other cancer tissues, and is significantly related to the degree of tumor infiltration, distant metastasis and differentiation. SUMMARY

[0005] The present application provides an anti-B7H3 protein monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1; and the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 2.

[0006] SEQ ID NO. 1:

[0007] QSVKESEGGLFKPTDTLTLTCTVSGFSLSRDAISWVRQAPGNGLEWIGIINSYGSTYYASWAKSRSTITRNTNENTVTLRMTSLTAADTATYFCARGALGAGTNIWGPGTLVTVSS

[0008] SEQ ID NO. 2:

[0009] QVLTQTPASVSEPVGGTVTIKCQASQSLYKNNYLSWYQQKPGQPPKLLVYYATTLASGVPSRFKGSGSGTEYTLTISDLECDDAATYYCAGDYGGGINGFGGGTEVVVK

[0010] Further, the coding DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO. 3, and the coding DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO. 4.

[0011] SEQ ID NO. 3:

[0012] CAGTCAGTGAAGGAGTCCGAGGGAGGTCTCTTCAAGCCAACGGATACCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTCGCGACGCAATAAGCTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAATGGATCGGGATCATTAATAGTTATGGTAGCACATACTACGCGAGCTGGGCGAAAAGCCGATCCACCATCACCAGAAACACCAACGAGAACACGGTGACTCTGAGAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGAGGGGCTCTTGGTGCTGGTACTAACATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0013] SEQ ID NO. 4:

[0014] CAAGTGCTGACCCAGACTCCAGCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGTCTTTATAAGAACAACTACTTGTCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGGTCTATTATGCAACCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACACTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGATTATGGTGGTGGTATTAATGGTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA

[0015] Further, the monoclonal antibody specifically recognizes the B7H3 protein.

[0016] Further, the monoclonal antibody is a rabbit monoclonal antibody.

[0017] Further, the clone number of the rabbit monoclonal antibody is 97A8.

[0018] The inventor also provides a method for preparing an anti-B7H3 protein monoclonal antibody, and the antigen for immunizing the rabbit is a recombinant protein, which is expressed by E. coli.

[0019] Further, the recombinant protein comprises a B7H3 protein fragment and a HIS protein tag.

[0020] Further, the B7H3 protein fragment is a fragment of 29-245 sites, which is the amino acid sequence shown in SEQ ID NO. 5.

[0021] SEQ ID NO. 5:

[0022] LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRV

[0023] ADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQG

[0024] LFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFP

[0025] Further, the plasmid vector selected in the recombinant process is pGEX-4T-AB1.

[0026] The inventor also provides a B7H3 protein immunological detection reagent containing the above-mentioned anti-B7H3 protein monoclonal antibody as an effective component.

[0027] Differing from the prior art, the beneficial technical effects of the present application are: the above-mentioned technical solution provides an anti-B7H3 protein rabbit monoclonal antibody, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1; the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 2. The antibody has high specificity and sensitivity, can specifically recognize cells expressing B7H3 protein, and is suitable for immunological detection, especially immunohistochemical detection. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Comparison of lung adenocarcinoma immunohistochemical staining results Figure 1 (Right is the rabbit monoclonal antibody B7H3 of the present application, and left is the commercially available B7H3).

[0029] Figure 2 Comparison of tonsil immunohistochemical staining results Figure 2 (Right is the rabbit monoclonal antibody B7H3 of the present application, and left is the commercially available B7H3). DETAILED DESCRIPTION

[0030] In order to describe the possible application scenarios, technical principles, specific schemes that can be implemented, and the purposes and effects that can be achieved of the present application in detail, the following will be described in detail in combination with the specific embodiments listed and with the aid of the drawings. The embodiments described in this paper are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, and cannot limit the protection scope of the present application.

[0031] In this paper, the term "embodiment" means that the specific features, structures or characteristics described in combination with the embodiments can be included in at least one embodiment of the present application. The term "embodiment" appearing at various positions in the specification does not necessarily refer to the same embodiment, and does not particularly limit the independence or association between other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, each technical feature mentioned in each embodiment can be combined in any way to form a corresponding implementable technical solution.

[0032] Unless otherwise defined, the meanings of technical terms used in the present application are the same as commonly understood by one of ordinary skill in the art to which the present application belongs; the use of related terms in the present application is only for the purpose of describing specific embodiments and is not intended to limit the present application.

[0033] In the description of the present application, the phrase "and / or" is a description of the logical relationship between objects, which means that there can be three relationships, for example, A and / or B, which means that there are three cases: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in the present application generally represents that the associated objects before and after are a "or" logical relationship.

[0034] In the present application, the phrases such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual quantity, primary and secondary or order relationship between the entities or operations.

[0035] In the present application, the phrases "including", "containing", "having" or other similar expressions used in the sentences are intended to cover non-exclusive inclusion, and these expressions do not exclude the presence of additional elements in the process, method or product including the described elements, so that the process, method or product including a series of elements can not only include those limited elements, but also include other elements not explicitly listed, or also include elements inherent to such process, method or product.

[0036] In the present application, "greater than", "less than", "exceed" and other expressions are understood as not including the number, and "above", "below", "within" and other expressions are understood as including the number. In addition, in the description of the embodiments of the present application, the meaning of "multiple" is more than two (including two), and similar expressions related to "multiple" are also understood in this way, for example, "multiple groups", "multiple times" and the like, unless otherwise explicitly limited.

[0037] Preparation of recombinant B7H3 protein fragment of Example 1

[0038] I. Gene optimization and synthesis

[0039] According to the protein sequence with accession number Q5ZPR3 in Uniprot database, the protein fragment of 29-245 sites of B7H3 is selected, which is directly optimized into a gene fragment suitable for expression in E. coli Rosetta (DE3), and cloned into pGEX-4T-AB1 vector.

[0040] The E. coli Rosetta (DE3) competent cells were transformed with the expression vector, and the clones on the plate were inoculated and subjected to bacterial liquid PCR identification. The clones with positive PCR results were selected for sequencing analysis, and the clones with completely correct sequences were used.

[0041] Different antigens were selected for immunization, and different antibodies with different binding properties were prepared. The molecule contains multiple variants caused by variable splicing, which ultimately leads to different recognition abilities and patterns of different antibodies to cells expressing the antigen. According to the published sequence, the B7H3 molecule was analyzed, and based on the structure, antigenicity, hydrophilicity and hydrophobicity of the constituent amino acids, and the secondary structure, a suitable soluble expression region with good immunogenicity was selected for recombinant expression. The gene sequence of the 29-245 site of B7H3 was selected for codon optimization, and the molecular weight was about 70-75 kDa. The B7H3 protein was obtained by designing the prokaryotic expression gene sequence through sequence optimization. The recombinant immunogen is composed of the B7H3 protein fragment with antigenicity and the protein tag for purification of the recombinant protein. The protein tag is HIS.

[0042] II. Protein expression and purification

[0043] The single colony culture overnight bacteria were transferred to 100 mL LB medium at a ratio of 1:100, and ampicillin was added at a final concentration of 100 μg / mL. The culture was incubated at 37°C with shaking until the OD600 was 0.6-0.8. Then, 0.8 mmol / L IPTG was added, and the culture was incubated at 37°C with shaking for 4 h. After harvesting the bacteria, ultrasonic disruption was performed. The recombinant protein has a histidine tag, and nickel column was used for affinity purification of the protein. Elution was performed with 150 mmol / L imidazole, and SDS PAGE separation and detection were performed. The pGEX-4T-AB1-B7H3 (29-245 aa) was expressed in the supernatant, and the protein concentration after purification was 1 mg / mL, and the purity reached 85%, meeting the requirements for animal immunization and antibody screening and identification.

[0044] I. Immunization and ELISA detection

[0045] The recombinant B7H3 protein in Example 1 was emulsified with Freund's complete adjuvant, and 6 rabbits were selected for immunization at a dose of 300 μg / rabbit. Booster immunization was performed once at 21, 28, and 49 days, respectively, and the antigen was emulsified with Freund's incomplete adjuvant at a dose of 150 μg / rabbit. After the third immunization, serum ELISA titer detection was performed, and the A rabbit had an immune titer greater than 1:2.56K, and the immune effect met the requirements. The serum of 4 rabbits after the fourth immunization was subjected to IHC detection, and according to the results (i.e., using the positive control photo corresponding to the antibody for detection, and observing immunohistochemical staining at the corresponding detection site in the positive control photo), 1 rabbit (number: A) was selected for subsequent monoclonal antibody screening.

[0046] II. Spleen cell isolation and B lymphocyte sorting

[0047] Single antibody preparation was performed on selected rabbits. Spleens were removed 3 days after the booster immunization, and the rabbit spleens were placed in RMPI base medium containing 100 U / ml penicillin and 100 ug / ml streptomycin, cut into pieces with a surgical blade and transferred to a 100 pm cell sieve for grinding. The obtained cell suspension was filtered to remove large cell clumps and tissue envelopes, and after centrifugation at 400 g for 5 minutes, the supernatant was removed and the spleen cell clumps were retained. The spleen cell clumps were resuspended with a hypotonic solution and the red blood cells were lysed, and then centrifuged at 400 g for 5 minutes, and the spleen cells were retained. The spleen cells were resuspended with RMPI base medium containing 100 U / ml penicillin and 100 ug / ml streptomycin, centrifuged at 400 g for 5 minutes, and the obtained spleen cells were resuspended with complete medium (RMPI base medium containing 10% fetal bovine serum, 100 U / ml penicillin and 100 ug / ml streptomycin) for standby.

[0048] The specific steps of B lymphocyte sorting are described in Chinese Patent 201910125091.4 "Method for efficiently isolating single antigen-specific B lymphocytes from spleen cells".

[0049] About 2000 single B cell clones were sorted and cultured, and positive clones specifically recognizing the recombinant B7H3 protein in Example 1 were screened by ELISA. According to the ELISA data, 50 supernatants from high to low were selected for IHC verification.

[0050] III. Detection of positive clones of B cell culture

[0051] Single B cell culture supernatants were verified by IHC using a multi-tumor tissue chip and a normal tissue chip containing B7H3 positive and negative. The IHC results of the culture supernatants of 8 clones were better, and the LEM supernatant was prepared for IHC verification to determine the clone (97A8) with excellent sensitivity and specificity.

[0052] The screening criteria for sensitivity and specificity are as follows: the positive control photograph corresponding to the antibody is used for detection, and immunohistochemical staining can be observed on the corresponding detection site in the positive control photograph, while no immunohistochemical staining is observed on the non-detection site. Compared with the control antibody, the staining intensity is even higher than that of the control antibody.

[0053] IV. Cloning of rabbit monoclonal antibody gene (97A8) and construction of rabbit monoclonal antibody expression plasmid

[0054] Positive clones were harvested, lysed, and RNA was extracted and reverse-transcribed into cDNA. Using PCR, naturally paired rabbit monoclonal antibody light and heavy chain variable region genes were amplified from the cDNA of the corresponding positive clones and sequenced. Rabbit monoclonal antibody expression vector plasmids were constructed using the naturally paired rabbit monoclonal antibody light and heavy chain variable region gene sequences.

[0055] Example 2 was completed by Fuzhou Maixin Biotechnology Development Co., Ltd. on behalf of Dima Biotechnology (Wuhan) Co., Ltd., and Fuzhou Maixin Biotechnology Development Co., Ltd. performed the immunohistochemical screening.

[0056] Example 3B7H3 rabbit monoclonal antibody expression

[0057] 1. Plasmid Amplification and Extraction

[0058] Take out a tube (100 μl) of competent bacteria (DH5α), insert it into ice, and ice bath for 5-10 minutes; add 5 μl of plasmid and shake gently, then place on ice for 30 minutes; shake gently and place in a 42°C water bath for 90 seconds for heat shock, then quickly return to ice and let it stand for 5 minutes; add 800 μl of LB culture medium (Note: does not contain antibiotics) to the above in the clean bench, mix gently, fix on a shaker and shake at 37°C for 1 hour; take 50-100 μl of the above transformation mixture in the clean bench, add it dropwise to the marked solid LB flat plate culture dish containing Amp, and spread it evenly with a glass coating rod (sterilized); first place it upright in a 37°C incubator for 30 minutes to allow the bacterial liquid on the surface to completely penetrate into the culture medium, and then invert it and place it in a 37°C incubator for overnight culture. Use a pipette to pick up a single colony and inject it into 4 ml LB medium (containing 2 uL 200 mg / ml Amp), and culture it in a shaking incubator at 37°C for 16 h at 37°C and 220 rpm.

[0059] 1 ml of bacterial solution was added to 100 ml of LB medium (containing 50 μl of 200 mg / ml Amp) and cultured at 37°C in a shaking incubator for 16 h. Plasmids were extracted using the SanPrep Endotoxin-Free Plasmid Mini-Amount DNA Extraction Kit (Sanggong) according to the manufacturer's instructions.

[0060] 2. Transfection

[0061] Adjust the concentration of 293F cells to 2.5-3 × 10 cells / mL using Expi293F™ Expression Medium. 6 viable cells / ml and culture overnight.

[0062] 1) Count the viable cells using a hemocytometer; the concentration of viable cells is approximately 4.5-5.5×10 6 viable cells / ml, and the number of viable cells must meet the requirements of the expression system;

[0063] 2) Dilute cell concentration to 3 x 105viable cells / ml with Expi293FTM Expression Medium 6 viable cells / ml;

[0064] 3) Add plasmid DNA to Opti-MENTM I Reduced Serum Medium, gently flick, invert to mix;

[0065] 4) Gently invert ExpiFectamine TM 293 Reagent 4-5 times, mix ExpiFectamine TM 293 Reagent and Opti-MENTM I Reduced Serum Medium, gently flick and invert 2-3 times, let stand at room temperature for 5 min;

[0066] 5) Mix solutions from steps 3) and 4), gently flick and invert 2-3 times, mix well;

[0067] 6) Let solution from step 5) stand at room temperature for 10-20 min;

[0068] 7) Slowly pipette mixture into cell culture, gently rock flask;

[0069] 8) Incubate at 37°C, 8% CO2, with shaking for 5-7 days;

[0070] 9) After 18-22 hours, add ExpiFectamine TM 293 Transfection Enhancer1 and ExpiFectamine TM 293 Transfection Enhancer2 (Note: mix ahead of time before use), and gently rock to mix, continue incubation.

[0071] III. Purification of Monoclonal Antibodies

[0072] Purify antibodies from supernatant using HiTrap rProtein A FF affinity chromatography according to manufacturer's instructions. Determine purity by SDS-PAGE gel and concentration by Bradford method. Store purified antibodies at -20°C.

[0073] Example 4. Immunohistochemistry Tissue Chip Staining and Identification

[0074] I. Chip Preparation Procedure

[0075] HE staining was performed on each sample to determine the tumor site. The tumor target site was circled and prepared for punching. When making the blank recipient wax block, a plastic frame was placed on the mold, and melted wax (melting point at 55-58°C) was poured into the mold. After cooling to room temperature, the mold was placed in a -20°C refrigerator for 6 min, and the wax block was removed from the mold. On the tissue sample machine, a 1 mm diameter sample needle was selected to punch a hole in the recipient wax block, with a hole depth of 3-4 mm. Another 1 mm diameter punching needle was used to punch a hole in the marked site of the wax block to collect a tissue core, with a length of about 0.1 mm shallower than the hole depth of the recipient wax block. The collected tissue core was directly inserted or carefully clamped with tweezers into the empty hole of the recipient wax block. This was repeated until all sample points were prepared. Finally, a glass slide was used to flatten all the tissue cores, making the tissue core block flat and smooth. The prepared tissue core block was placed in the wax block making mold and placed in a 60°C oven for 15 min to fuse the tissue core with the wax of the recipient wax block. Then the mold was gently removed from the oven, and the semi-melted wax was allowed to cool at room temperature for about 30 min. The tissue core block was then placed in a -20°C refrigerator for 6 min, and then removed from the mold. The tissue core block was sectioned or stored in a 4°C refrigerator for future use. After trimming, continuous sections were cut with a thickness of 3 μm. The continuous sections were floated in cold water to naturally expand, and then transferred to warm water at 45°C for 30 seconds. The sections were mounted on slides treated with polylysine, and the prepared tissue core block was placed in a 65°C oven for 2 hours. After cooling to room temperature, the tissue core block was stored in a -4°C refrigerator.

[0076] II. IHC Staining and Analysis

[0077] Routine xylene de-waxing was performed 3 times for 6 minutes each, with hydration in 100%, 100%, 95%, and 85% ethanol for 3 minutes each, and finally rinsed with tap water. Antigen retrieval was performed, and then the sections were placed in a wet box and rinsed with PBS for 3 x 3 minutes. 3% H2O2 was added and incubated for 10 minutes, and then rinsed with PBS for 3 x 3 minutes. The sections were spun dry, and the appropriate dilution of primary antibody was added (the first dilution was designed according to the concentration of the antibody). The sections were incubated at room temperature (25°C) for 1 hour, rinsed with PBS for 3 x 3 minutes, and then the secondary antibody was added and incubated at room temperature for 15-30 minutes. The sections were rinsed with PBS for 3 x 3 minutes, spun dry, and then developed with freshly prepared DAB developing solution for 3-10 minutes. Hematoxylin counterstaining was performed for 25 seconds, and then rinsed with PBS for 30 seconds. The sections were dehydrated in an alcohol gradient of 85% (3 minutes), 95% (3 minutes), 100% (3 minutes), and 100% (3 minutes), and finally cleared with xylene for 3 minutes. The sections were mounted with neutral balsam.

[0078] The immunohistochemical staining results are divided into positive and negative. Positive expression must be at the site of cell and tissue-specific antigens to be considered positive. In the case of clear distribution of tissue staining and accurate cell localization, the staining results are further divided according to the difference in staining intensity, as follows:

[0079] 1. The sample is weakly positive; marked as "+".

[0080] 2. The sample is moderately positive; marked as "++".

[0081] 3. The sample is highly positive; marked as "+++".

[0082] 4. The sample is negative, marked as "-".

[0083] III. Data Statistics

[0084] 1. Tumor tissue chip detection results:

[0085] The antibody B7H3 (97A8) and the commercially available antibody B7H3 (rabbit polyclonal antibody) were used to simultaneously detect 82 cases of lung adenocarcinoma and compare the detection results. The immunohistochemical results of B7H3 were statistically analyzed. The entire test process adopted a double-blind design, and the statistical results are as follows:

[0086]

[0087] The results show that the rabbit monoclonal antibody B7H3 (97A8) has accurate staining localization, clear staining, no non-specific staining, and clean background. In immunohistochemical detection, the positive rate and positive intensity are higher than those of the commercially available antibody. There are 2 cases of positive detection of the B7H3 rabbit monoclonal antibody 97A8 prepared by the present application, while the detection result of the commercially available antibody is negative, indicating that the sensitivity of the rabbit monoclonal antibody B7H3 (97A8) is higher than that of the commercially available antibody.

[0088] Figure 1 Figure 1 is a comparison of immunohistochemical staining results of a lung adenocarcinoma (the right is the rabbit monoclonal antibody B7H3 of the present application, and the left is the commercially available B7H3).

[0089] 2. Normal tissue chip detection results:

[0090] The normal tissue chip includes 30 normal tissue samples, which are mainly selected from fresh and timely fixed surgical specimens. Each type of tissue includes 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectum, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (infant), skin, bone marrow, peripheral nerve, lung, mesothelial cells.

[0091] The rabbit monoclonal antibody B7H3 (97A8) and the commercially available B7H3 antibody were synchronously detected on a normal tissue chip, and the positive and negative detection results were consistent, indicating that the specificity of the antibody in normal tissues was equivalent to that of the commercially available antibody.

[0092] Figure 2 A comparison chart of tonsil immunohistochemical staining results (the right is the rabbit monoclonal antibody B7H3 of the application, and the left is the commercially available B7H3).

[0093] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of the present application, the patent protection scope of the present application should not be limited. Any technical solution obtained by replacing or modifying the equivalent structure or equivalent process based on the essential concept of the present application, using the content described in the specification and drawings of the present application, and directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, etc., are all included in the patent protection scope of the present application.

Claims

1. An anti-B7H3 protein monoclonal antibody, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

2.

2. The monoclonal antibody according to claim 1, characterized in that, The coding DNA sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 3, and the coding DNA sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

4.

3. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody specifically recognizes B7H3 protein.

4. The monoclonal antibody of claim 1, wherein, The monoclonal antibody is a rabbit monoclonal antibody.

5. A B7H3 protein immunoassay reagent, characterized by, The immunoassay reagent contains the anti-B7H3 protein monoclonal antibody of claim 1 as an effective component.

6. The immunoassay test reagent of claim 5, wherein, The immunoassay includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

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