Anti-ERBB2 monoclonal antibodies, their preparation methods and applications

Abstract

This invention discloses an anti-ERBB2 monoclonal antibody, its preparation method, and its applications, belonging to the field of biomedical technology. This anti-ERBB2 monoclonal antibody uses the intracellular domain of the human ERBB2 protein as an immunogen and is obtained through animal immunization, B cell screening, gene cloning, and recombinant expression techniques. It can specifically recognize the human ERBB2 protein, exhibiting high affinity and high specificity, and can recognize both native and denatured ERBB2 proteins. This monoclonal antibody can be applied in immunohistochemistry, immunofluorescence, Western blotting (WB), flow cytometry, ELISA detection, and tumor targeted therapy. Experimental results show that this antibody has low background and no non-specific binding, accurately detecting ERBB2-overexpressing tumor cells and tissues, and has significant application value in the diagnosis, prognostic assessment, and targeted therapy of ERBB2-related diseases such as breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to anti-ERBB2 monoclonal antibodies, their preparation methods, and applications. Background Technology

[0002] Human epidermal growth factor receptor 2 (HER2, ERBB2) is a member of the epidermal growth factor receptor (EGFR / ErbB) family and is a transmembrane tyrosine kinase receptor. ERBB2 gene amplification or protein overexpression is commonly found in various malignant tumors, including breast cancer, gastric cancer, ovarian cancer, and non-small cell lung cancer, and is closely associated with high tumor invasiveness, poor prognosis, and high recurrence rate. Therefore, ERBB2 is not only an important prognostic biomarker for tumors but also a key target for anti-tumor targeted therapy. Currently, monoclonal antibody drugs targeting ERBB2 (such as trastuzumab and pertuzumab) are used clinically for treatment, but in practice, problems such as primary drug resistance, secondary drug resistance, or significant side effects still exist in some patients.

[0003] In the field of pathological diagnosis, existing detection antibodies still have room for improvement in terms of sensitivity, specificity, and binding ability to antigens under different fixation conditions. For example, some antibodies have high background in immunohistochemical detection or insufficient ability to recognize antigenic epitopes in formalin-fixed paraffin-embedded (FFPE) tissues, leading to false negatives or difficulties in interpreting results.

[0004] Therefore, developing novel anti-ERBB2 monoclonal antibodies with higher affinity, higher specificity, the ability to recognize new epitopes, and applicability to multiple detection platforms and potential therapeutic applications is of great clinical significance and application value for improving the accuracy of tumor diagnosis and developing new treatment options. Summary of the Invention

[0005] 1. Purpose of the invention The purpose of this invention is to provide an anti-ERBB2 monoclonal antibody, its preparation method, and its applications. This anti-ERBB2 monoclonal antibody is obtained by immunizing New Zealand white rabbits with the intracellular polypeptide of the ERBB2 protein as an immunogen, followed by cell sorting and other steps. It can specifically recognize the ERBB2 protein. Immunohistochemical (IHC) detection in various tissues has shown that this anti-ERBB2 monoclonal antibody can effectively detect the expression of ERBB2 protein on cells. It can be applied to the detection and screening of ERBB2 protein expression using techniques such as immunohistochemistry, indirect ELISA, immunofluorescence, and flow cytometry.

[0006] 2. Technical Solution To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: As a first aspect of the present invention, the present invention provides an anti-ERBB2 monoclonal antibody or an antigen-binding fragment thereof, the anti-ERBB2 monoclonal antibody being specifically capable of binding to the ERBB2 protein, comprising a heavy chain and a light chain: The amino acid sequence of the heavy chain is shown in SEQ ID NO.4; the amino acid sequence of the light chain is shown in SEQ ID NO.5.

[0007] Furthermore, the above-mentioned anti-ERBB2 monoclonal antibody or its antigen-binding fragment, wherein the antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab)'2 fragment, a single-chain Fv protein (scFv), or a disulfide-stabilized Fv protein (dsFv).

[0008] As a second aspect of the invention, the invention also provides a nucleic acid that encodes the above-mentioned anti-ERBB2 monoclonal antibody or its antigen-binding fragment.

[0009] Furthermore, the aforementioned nucleic acids include: The nucleotide sequence shown in SEQ ID NO.2 is used to encode the heavy chain of the above-mentioned amino acid sequence shown in SEQ ID NO.4; the nucleotide sequence shown in SEQ ID NO.3 is used to encode the light chain of the above-mentioned amino acid sequence shown in SEQ ID NO.5.

[0010] As a third aspect of the present invention, the present invention also provides a recombinant expression vector containing the above-mentioned nucleic acid, which can express an anti-ERBB2 monoclonal antibody or its antigen-binding fragment.

[0011] Furthermore, the recombinant expression vector includes a pcDNA3.1 plasmid containing the aforementioned nucleic acid.

[0012] As a fourth aspect of the present invention, the present invention also provides a recombinant expression cell comprising the above-described recombinant expression vector or the above-described nucleic acid, which can express an anti-ERBB2 monoclonal antibody or its antigen-binding fragment.

[0013] Furthermore, the aforementioned recombinant expression cells include the HEK293 cell line.

[0014] As a fifth aspect of the present invention, the present invention also provides the use of the above-mentioned nucleic acid, recombinant expression vector, and recombinant expression cell in the preparation of anti-ERBB2 monoclonal antibody or its antigen-binding fragment.

[0015] Furthermore, the above applications include: Recombinant expression cells were obtained by transfecting cells with the above-mentioned recombinant expression vector and then cultured; or the above-mentioned recombinant expression cells were cultured directly. After culturing, the supernatant was collected and purified to obtain anti-ERBB2 monoclonal antibody or its antigen-binding fragment.

[0016] As a sixth aspect of the present invention, the present invention also provides a method for preparing the above-mentioned anti-ERBB2 monoclonal antibody or its antigen-binding fragment, comprising: Recombinant expression cells were obtained by transfecting cells with the above-mentioned recombinant expression vector and then cultured; or the above-mentioned recombinant expression cells were cultured directly. After culturing, the supernatant was collected and purified to obtain anti-ERBB2 monoclonal antibody or its antigen-binding fragment.

[0017] As a seventh aspect of the present invention, the present invention also provides the application of the above-mentioned anti-ERBB2 monoclonal antibody or its antigen-binding fragment, nucleic acid, recombinant expression vector, and recombinant expression cell in the detection of ERBB2 protein or the preparation of products for detecting ERBB2 protein.

[0018] Furthermore, the above-mentioned detection of ERBB2 protein includes any one or more of the following methods: immunohistochemistry, ELISA, immunofluorescence, and flow cytometry.

[0019] Furthermore, the aforementioned ERBB2 protein detection products include any one or more of the following: kits, antibody conjugates, antibody chips, etc.

[0020] As an eighth aspect of the present invention, the present invention also provides a kit for detecting ERBB2 protein, the kit comprising the above-mentioned anti-ERBB2 monoclonal antibody or its antigen-binding fragment, wherein the anti-ERBB2 monoclonal antibody or its antigen-binding fragment is used as the primary antibody.

[0021] Furthermore, the above-mentioned kit is a flow cytometry detection kit, including the aforementioned anti-ERBB2 monoclonal antibody or its antigen-binding fragment, and also includes flow cytometry detection reagents. Even further, the above-mentioned flow cytometry detection kit also includes: fluorescently labeled secondary antibody, PBS, bovine serum albumin, Human IgG, 7-AAD, and other reagents.

[0022] Furthermore, the above-mentioned kit is an immunohistochemical detection kit, comprising the aforementioned anti-ERBB2 monoclonal antibody or its antigen-binding fragment, and also including immunohistochemical detection reagents. Even further, the above-mentioned immunohistochemical detection kit also includes: HRP-labeled secondary antibody, EDTA retrieval solution, catalase blocking solution, DAB concentrate, DAB buffer, hematoxylin, and blueing solution, etc.

[0023] As a ninth aspect of the present invention, the present invention also provides the application of the above-described kit for detecting ERBB2 protein in the detection of ERBB2 protein.

[0024] Specifically, the above-mentioned kit for detecting ERBB2 protein is a flow cytometry detection kit, and its applications include: cell resuscitation, FC receptor blockade, primary antibody incubation, secondary antibody incubation, 7-AAD activity staining, and data collection.

[0025] Specifically, the above-mentioned kit for detecting ERBB2 protein is an immunohistochemical detection kit, and its applications include: dewaxing, antigen retrieval, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, DAB staining, counterstaining, dehydration, mounting, and microscopic examination.

[0026] 3. Technical Effects Compared with the prior art, the advantages of this invention are as follows: (1) The anti-ERBB2 monoclonal antibody or its antigen-binding fragment provided by the present invention is obtained by immunizing New Zealand white rabbits with the intracellular polypeptide of ERBB2 protein as an immunogen and by cell sorting and other steps. It includes a heavy chain with an amino acid sequence as shown in SEQ ID NO.4 and a light chain with an amino acid sequence as shown in SEQ ID NO.5. It can specifically recognize and detect the expression of ERBB2 protein on tumors or cancer tissues such as breast cancer, gastric cancer, and ovarian cancer, which is beneficial to obtaining accurate evaluation and detection results.

[0027] (2) The anti-ERBB2 monoclonal antibody or its antigen-binding fragment and its preparation method and application provided by the present invention have a shorter cycle and are not limited by myeloma cells compared with traditional hybridoma methods; compared with phage display technology, natural antibodies have higher affinity. Attached Figure Description

[0028] Figure 1 These are the immunohistochemical results of anti-ERBB2 specific polyclonal antibody serum in human breast cancer tumor tissue. From left to right, these are the immunohistochemical results of serum obtained from rabbits with immunogen immunization numbers K180, K181, and K182 in human breast cancer tumor tissue.

[0029] Figure 2 The images show the immunohistochemical results of the supernatant from the anti-ERBB2 clone screening stage of single B cell expansion culture in human breast cancer tumor tissue and normal human breast cancer tissue. The left image shows human breast cancer tumor tissue, and the right image shows normal human breast cancer tissue.

[0030] Figure 3 These are the immunohistochemical results of anti-ERBB2 monoclonal antibody in human breast cancer tumor tissue and normal human breast cancer tissue. The left image is human breast cancer tumor tissue; the right image is normal human breast cancer tissue.

[0031] Figure 4These are the results of immunoblotting detection of anti-ERBB2 monoclonal antibody in MCF7 and SKBR-3 cells, where: Lane 1 is MCF7; Lane 2 is SKBR-3. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments.

[0033] It should be noted that, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0034] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0035] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable. As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0036] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as "less than about 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0037] In this invention, unless otherwise defined, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, scFv, diabetic bodies, single-domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), probodies, and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0038] The monoclonal antibodies of this invention can be prepared using a variety of techniques, such as hybridoma techniques (see, for example, Kohler et al., Nature, 256:495, 1975), recombinant DNA techniques (see, for example, U.S. Patent Application 4,816,567), or phage antibody library techniques (see, for example, Clackson et al., Nature, 352:624-628, 1991, or Marks et al., J. Mol. Biol. 222:581-597, 1991). The antibodies can be purified using known techniques, such as affinity chromatography with protein A or protein G. Subsequently, or alternatively, the specific antigen (the target molecule recognized by the antibody) or its epitope can be immobilized on a column, and the immunospecific antibody can be purified by immunoaffinity chromatography. For purification of immunoglobulins, please refer to, for example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0039] Example 1 This embodiment provides the design and synthesis of the ERBB2 polypeptide immunogen, specifically including: Using ERBB2 (Uniprot ID: P04626) as the target antigen, the ERBB2 protein sequence was analyzed. The structure, linearity, length, and hydrophilicity / hydrophobicity of the ERBB2 protein sequence were predicted. Finally, the region peptide aa1244-aa1252 was selected as the immunogen for preparing anti-ERBB2 monoclonal antibody. This peptide is an intracellular region, which is more suitable for the development of immunohistochemical applications, etc. Its specific sequence is: ENPEYLGLD (SEQ ID NO.1).

[0040] Simultaneously, three amino acids, EEC, were added to the C-terminus of peptide aa1244-aa1252 to crosslink KLH, named peptide A, for animal immunization; and a biotin was added to the N-terminus of peptide aa1244-aa1252 to crosslink avidin magnetic beads, named peptide B.

[0041] Both polypeptides were synthesized by Anhui Guoping Pharmaceutical Co., Ltd.

[0042] Example 2 This embodiment provides the screening and preparation of anti-ERBB2 monoclonal antibodies, specifically including the following steps: (1) Animal immunization and serum detection The polypeptide A synthesized in Example 1 was conjugated with KLH as an immunogen. 500 μg of the immunogen was mixed with 0.5 mL of complete Freund's adjuvant and emulsified. Three New Zealand white rabbits (numbered K180, K181, and K182) were immunized subcutaneously at multiple sites on the back. Fifteen days later, 0.25 mL of incomplete Freund's adjuvant and 250 μg of the immunogen were mixed and emulsified for a second immunization. A third immunization was performed 15 days later, using the same method as the second. A fourth immunization was performed 15 days later, using the same method as the second. Ten days after the final immunization, blood was collected from the marginal ear vein of the rabbits. Antiserum was obtained by centrifugation at 3000 rpm for 20 min. Serum antibody titers were measured using indirect ELISA to assess the antigen immunization effect.

[0043] Indirect ELISA detection of serum antibody titers included: ERBB2 peptide A was diluted to 0.5 μg / mL with carbonate buffer (50 mM, pH 9.6), and 100 μL / well of peptide A was coated onto an ELISA plate and incubated overnight at 4°C; the next day, the plate was blocked with 2‰ gelatin at 37°C for 1 h. Rabbit antiserum was serially diluted as the primary antibody, incubated at 37°C for 1 h, followed by 1:10000 goat anti-rabbit secondary antibody, incubated at 37°C for 1 h, and then chromogenic reagent was added for color development. The absorbance at OD450 was read using an ELISA reader. The results are shown in Table 1. The OD450 value of rabbit serum from rabbit K181 at a dilution of 1:64000 was higher than that of the other two rabbits. Lymphocytes were obtained from the spleen of New Zealand white rabbit K181 after grinding.

[0044] Table 1. Titer of anti-ERBB2 specific polyclonal antibody serum at different dilutions

[0045] Immunohistochemical analysis of rabbit anti-ERBB2 specific polyclonal antibody serum showed membrane staining in human breast cancer tumor tissue. Figure 1 ).

[0046] (2) Specific B cell enrichment and proliferation Under aseptic conditions, the spleen of rabbit K181 was removed, ground in a 10 cm culture dish, and passed through a cell sieve to obtain B lymphocytes. The obtained B lymphocytes were then resuspended and diluted to a density of 102. 5 The streptavidin magnetic beads used in this invention were purchased from Suzhou Weidu Biotechnology Co., Ltd., catalog number CMP1001SB. 25 μL of magnetic beads were washed with PBS and then directly cross-linked with 100 μg of polypeptide B to obtain a magnetic bead mixture; the lymphocytes obtained from the spleen were counted and then placed in a 5×10⁻⁶ saturated solution. 6 One live cell was transferred to a 9 cm culture dish and incubated on a shaker at 37°C for 2 h. The cell and magnetic bead mixture was then transferred to a 15 mL centrifuge tube and placed on a Thermo DynaMag™-15 magnetic rack (catalog number: 12301D). The supernatant was discarded, and the cells were resuspended in fresh 2% FBS 1640 medium. Washing continued until no cells were visible in the supernatant. The specific B cells sorted by the magnetic beads were resuspended in culture medium and counted. After counting, the cells were diluted, plated in 96-well plates, and incubated at 37°C for 7 days in a 5% CO2 incubator for proliferation.

[0047] (3) Cloning screening After proliferation, a small population of B cells capable of efficiently secreting monoclonal antibodies was obtained from 96-well plates. Antigen-specific clones were then obtained using an indirect ELISA method. The experimental method is as follows: 1) Take peptide A from the freezer and prepare a coating buffer solution at a concentration of 0.5 µg / mL using carbonate. Add the prepared coating solution at a rate of 50 µL / well to the labeled 96-well microplate. Gently shake the plate to spread the coating solution to the bottom of the wells. Apply adhesive tape to the top 96-well microplate and incubate overnight at 2-8°C. Shake off the coating solution the next day. 2) Sealing: Add 60 µL of 1% BSA to each well and let stand at room temperature for 45 min to seal; 3) Washing: Discard the blocking solution and wash once with 200 µL of 1×PBST per well. 4) Sample addition: Add 50 μL of supernatant to each well using a multi-channel pipette; 5) Add positive and negative controls: K181 four-time immunization rabbit serum, dilute the serum with 1% BSA 1:1000, mix well, and add to two wells in the last column of the plate. The negative control does not add primary antibody to the last two wells. 6) Washing: Discard the primary antibody and wash twice with 200 µL of 1×PBST. 7) Add secondary antibody: Fc-HRP 1:10000 dilution, 50 µL / well, incubate at room temperature for 40 min; 8) Washing: Discard the secondary antibody, wash twice with 200 µL of 1×PBST, and then pat dry; 9) Color development: Add 50 µL of TMB substrate to each well and develop the color at room temperature in the dark for 5 min; 10) Termination: Add 50 µL of 4.9% phosphoric acid to each well in the order of color development to terminate the reaction; 11) Reading: Read the plate at the OD450 wavelength of the microplate reader within 15 minutes after termination.

[0048] The supernatant of ELISA antigen-specific positive clones was selected for immunohistochemical verification, and the results were as follows: Figure 2 As shown, a B cell clone that can specifically recognize the ERBB2 protein in cancer tissue was obtained.

[0049] (4) Construction and expression of recombinant antibody vector Immunohistochemical staining was used to detect the specific recognition of ERBB2 protein in cancer tissue. B cells were lysed, and mRNA was obtained. RT-PCR was then used to obtain cDNA containing the heavy and light chain sequences specific to B cells. The obtained cDNA was constructed into the expression vector pCDNA3.1 (Beijing Qingke Biotechnology Co., Ltd.) via homologous recombination, and the heavy and light chain gene sequences were obtained through sequencing analysis. The heavy and light chain gene sequences were then paired, and the constructed eukaryotic expression vector was transfected into the HEK293 cell line using transfection reagents. The cell supernatant was collected.

[0050] (5) Antibody sequence HEK293 cell supernatant collected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) confirmed that the antibody expressed by the plasmid containing the following nucleic acid had a good immune response to the antigen, and finally a positive antibody sequence was obtained.

[0051] The nucleic acid (H-DNA) sequence encoding the antibody heavy chain is as follows: The nucleic acid (L-DNA) sequence encoding the antibody light chain is as follows: (SEQ ID NO.3).

[0052] Based on the above nucleic acid sequences, the corresponding amino acid sequences of the heavy and light chains of the antibody were obtained: Antibody heavy chain (H) amino acid sequence: QSVEESGGRLVTPGTPLTLTCTVSGFSLSRYAMSWVRQAPGKGLEWIGIISSSGSTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARYSSDYTDFNIWGPGTL VTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCP PPELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKAR GQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ IDNO.4); Antibody light chain (L) amino acid sequence: QVLTQTPSSVSAAVGGTVTINCQSSQNVYSNNRLSWFQQKPGQPPKLLIYSASTLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGGYSGNINDSYAFGGGTEVVV KGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQID NO.5).

[0053] In this example, the confirmed positive expression vector was used to transfect a large number of cells. After culturing for 3-5 days, the cell suspension was collected, centrifuged, and the supernatant was taken. The cell supernatant was then purified by Protein A affinity chromatography. The purification steps are as follows: 1) Supernatant filtration: The cell culture supernatant was filtered through a 0.45 μm filter before incubation; 2) Sample loading: Calculate the required volume of Protein A packing material for incubation or sample loading based on the volume of the supernatant. Add the packing material to a 50 mL centrifuge tube and place it on a rotary shaker. Incubate at room temperature for 1 h. Then transfer the packing material to a 6 mL chromatography column. After incubation, let it stand at room temperature for 10 min to allow the packing material to settle. No standing is required after sample loading; subsequent washing can be performed directly. 3) Washing: Wash the packing material sequentially with 15 column volumes of Wash Buffer A and 1×PBS, measure the protein concentration of the post-column effluent, and elute when OD280 < 0.01; 4) Elution: Elution was performed using Elute Buffer B. Collection began when the OD280 of the eluent was >0.1 and continued until OD280 <0.1. 10×PBS was added to the neutralized eluent to bring it back to a 1×PBS environment. The IgG concentration was measured using a protein concentration analyzer (the absorbance of rabbit IgG needs to be manually set to 1.37), and the relevant data were recorded before ultrafiltration concentration.

[0054] After the above steps, rabbit anti-human ERBB2 monoclonal antibody with a purity greater than 95% can be obtained.

[0055] Example 3 This embodiment provides the application of the anti-ERBB2 monoclonal antibody screened by the present invention in the detection of ERBB2 protein.

[0056] In this embodiment, the detection method is immunohistochemistry, using anti-ERBB2 monoclonal antibody as the primary antibody and OneStep polymer HRP-GAR (Hangzhou Startech Biotechnology Co., Ltd., S0B4003) as the secondary antibody, specifically including: (1) The chip containing cancer tissue (mc) was sliced ​​with a thickness of 4 micrometers, spread in water at 42°C, and placed in a drying oven at 63~65°C for about 1 hour; (2) Dewaxing and hydration: The sections were placed in the following order: a) tissue clearing agent twice, 15 min each time; b) 100% alcohol twice, 5 min each time; c) 95% alcohol twice, 5 min each time; d) 85% alcohol once, 5 min each time; e) 75% alcohol once, 5 min each time. After the dewaxing and hydration, the sections were removed and rinsed with distilled water three times, 5 min each time (all operations were performed in a fume hood). (3) Antigen retrieval: Place the slide in a staining box containing sodium citrate buffer (prepared with 200 mL of deionized water, i.e., 4 mL of 50× retrieval solution) or EDTA retrieval solution. Add 2 L of distilled water to the antigen retrieval pot and place the staining box in a pressure cooker for high-temperature and high-pressure retrieval. After the pressure cooker starts to release gas, close the gas valve and continue for 20 min. Then stop heating, open the lid, take out the inner pot, and let it cool naturally. Rinse with distilled water 3 times and place on a shaker for 5 min each time. (4) Blocking endogenous peroxidase: After washing 3 times, use an immunohistochemical pen to draw circles on the slide. When drawing circles, make sure that the size of the tissue circles is about the same, spaced apart from each other, and the slide position is about in the middle of the circle. Block non-specific antigens with 10% sheep serum (prepared with PBS) at room temperature for 30 min. (5) Primary antibody addition: After blocking, knock off the blocking solution on the slide, add 80~100 μL of diluted primary antibody (anti-ERBB2 monoclonal antibody) per well, adjust according to the size of the circle, put the slide into the humidifier, and then slowly put it into the refrigerator at 4°C overnight. (6) Adding secondary antibody working solution: On the morning of the second day, take out the humidified box and let it stand at room temperature for 10 min; wash 3 times with 1×PBST, 5 min each time; add secondary antibody, incubate at room temperature for 30 min, and wash 3 times with PBST, 5 min each time; (7) DAB color development preparation: Based on 100 μL per well, 6 mL needs to be prepared, that is, 6 mL of color development buffer is added to 6 drops of color development solution; add freshly prepared color development solution to each slide, develop for 1~2 min, which can be observed with the naked eye, and the color development is stopped when a brownish-yellow color appears. After the time is up, rinse directly with tap water to stop the color development; counterstain with hematoxylin for 2~5 min; rinse with distilled water for 5 min, decolorize and return to blue in PBS for 30 s, and rinse with distilled water for 5 min; (8) Dehydration of slices: sequentially dehydrated and dried with gradient alcohols: a) 50% alcohol, 5 min; b) 75% alcohol, 5 min; c) 90% alcohol, 5 min; d) 100% alcohol 3 times, 5 min each time; e) xylene 2 times, 5 min each time; (9) Mounting: Add 50-100 μL of neutral resin to each slide, then slowly add a coverslip; (10) Slide scanning: After mounting, the slides are dried overnight. The next day, they are observed under a microscope and scanned with a tissue sectioner to obtain immunohistochemical results.

[0057] The results are as follows Figure 3 As shown, Figure 3The results of the detection of anti-ERBB2 monoclonal antibody in human breast cancer tumor tissue and normal human breast cancer tissue show that the antibody of the present invention can detect the expression of ERBB2 protein in human breast cancer tumor tissue, while staining is almost undetectable in normal tissue.

[0058] Example 4 This embodiment provides the application of the anti-ERBB2 monoclonal antibody screened by the present invention in the detection of ERBB2 protein.

[0059] In this embodiment, the detection method is Western blotting, which analyzes the specific binding of the purified antibody to the ERBB2 protein. Specifically, this includes: MCF7 and SKBR-3 cells in good culture condition were collected using SDS loading buffer to prepare cell lysis buffer (MCR7 was the negative control, and SKBR-3 was the positive material). SDS-PAGE electrophoresis was performed according to standard methods, and the target protein gel region was transferred to a PVDF membrane (activated with methanol) at a constant current of 250 mA for 120 min. After transfer, the PVDF membrane was blocked in 5% skim milk powder on a shaker at room temperature for 1 h. Anti-ERBB2 monoclonal antibody (0.5 mg / mL) was diluted 1:1000 as the primary antibody and incubated on a shaker at room temperature for 2 h. The membrane was washed 3 times with TBST for 10 min each time. HRP-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch, GPRB051801H) was added as the secondary antibody (1:10000) and incubated on a shaker at room temperature for 1 h. The membrane was washed 5 times with TBST for 10 min each time. The membrane was exposed to ECL chromogenic solution for imaging analysis in a luminescence imaging system.

[0060] Immunoblotting results as follows Figure 4 As shown, the anti-ERBB2 monoclonal antibody exhibits specific binding to the ERBB2 protein on Western blotting.

Claims

1. An anti-ERBB2 monoclonal antibody or its antigen-binding fragment, characterized in that, The anti-ERBB2 monoclonal antibody comprises a heavy chain and a light chain: the amino acid sequence of the heavy chain is shown in SEQ ID NO.4; the amino acid sequence of the light chain is shown in SEQ ID NO.

5.

2. The anti-ERBB2 monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The antigen-binding fragments include: Fab fragments, Fab' fragments, F(ab)'2 fragments, single-chain Fv proteins, or disulfide-stabilized Fv proteins.

3. A nucleic acid, characterized in that, The nucleic acid encodes the anti-ERBB2 monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that, The nucleic acid includes: the nucleotide sequence shown in SEQ ID NO.2 and the nucleotide sequence shown in SEQ ID NO.

3.

5. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid as described in claim 3 or 4.

6. A recombinant expression cell, characterized in that, The recombinant expression cell comprises the nucleic acid as described in claim 3 or 4, or the recombinant expression vector as described in claim 5.

7. The use of the nucleic acid of claim 3 or 4, or the recombinant expression vector of claim 5, or the recombinant expression cell of claim 6 in the preparation of anti-ERBB2 monoclonal antibody or its antigen-binding fragment.

8. The method for preparing the anti-ERBB2 monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The method includes: Recombinant expression cells are obtained by transfecting cells with the recombinant expression vector as described in claim 5, and the recombinant expression cells are cultured; or the recombinant expression cells as described in claim 6 are cultured. After culturing, the supernatant was collected and purified to obtain anti-ERBB2 monoclonal antibody or its antigen-binding fragment.

9. The use of the anti-ERBB2 monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2, or the nucleic acid as described in claim 3 or 4, or the recombinant expression vector as described in claim 5, or the recombinant expression cell as described in claim 6, in the preparation of products for detecting ERBB2 protein.

10. The application according to claim 9, characterized in that, The detection of ERBB2 protein includes any one or more of the following methods: immunohistochemistry, ELISA, immunofluorescence, and flow cytometry.

11. A kit for detecting ERBB2 protein, characterized in that, The kit comprises the anti-ERBB2 monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2.

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