Hybridoma cell strain 13-8-G2, anti-CD318 monoclonal antibody and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a hybridoma cell strain 13-8-G2, an anti-CD318 monoclonal antibody and application of the anti-CD318 monoclonal antibody. A light chain variable region of the anti-CD318 monoclonal antibody provided by the invention comprises a CDR1 as shown in SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 as shown in SEQ ID NO: 8; the heavy chain variable region comprises CDR1 as shown in SEQ ID NO: 11, CDR2 as shown in SEQ ID NO: 12 and CDR3 as shown in SEQ ID NO: 13, has higher affinity with CD318 protein, can specifically recognize the CD318 protein, has higher titer than a commercial antibody, and has important application prospects in preparation of a CD318 detection kit.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a hybridoma cell line 13-8-G2, an anti-CD318 monoclonal antibody and applications thereof. Background Art

[0002] CD318, also known as CUB domain protein 1 (CDCP1), is a cell surface glycoprotein composed of 836 amino acids, including an amino-terminal signal peptide containing 29 amino acid residues and an extracellular segment, a transmembrane segment, and an intracellular segment consisting of 636, 21, and 150 amino acids, respectively. The extracellular domain of CD318 contains three CUB domains and multiple glycosylation sites, and the intracellular small molecule domain contains multiple tyrosine residues that can be phosphorylated by Src family kinases (SFKs), of which Tyr734 is one of the most important tyrosine residues.

[0003] Several studies have shown that CD318 is significantly upregulated in a variety of malignant tumors, such as pancreatic cancer, renal cancer, liver cancer, breast cancer, lung cancer, colorectal cancer, prostate cancer, melanoma, etc. It is almost not expressed or lowly expressed in normal tissues, and its high expression is associated with tumor occurrence, development and poor prognosis. It has been reported that a variety of tumor-related proteins interact with CD318. CD318 is a key hub of multiple classical signaling pathways in tumor occurrence and progression, and its overexpression and activation are also associated with prognosis and drug resistance. Due to its important role in malignant tumors, CD318 has become a potential biomarker and therapeutic target for a series of cancers. Anti-CD318 monoclonal antibodies can be used for the detection, localization and functional studies of CD318, providing important tools and resources for tumor biology and clinical treatment.

[0004] As a key node of carcinogenic signaling, CD318 can be used as a tumor biomarker, and its detection can be used as a basis for diagnosing tumor development and treatment effects. CD318 can be used as a target for PET radiotracers for tumor imaging. In addition, CD318 and its mediated signaling pathways can also be used as targets for antibody drugs to treat cancer. In addition, CD318 can be used as a target for ADC drugs, and anti-CD318 antibodies can deliver cytotoxic payloads. At present, many pharmaceutical companies have made arrangements for the development of antibody drugs and ADC drugs targeting CD318, and these projects are all in the preclinical stage. The high expression of CD318 in a variety of tumor cells makes it suitable for the development of antibody drugs, ADC, CAR-T and other therapies. Combined with its application as a marker for tumor development and prognosis detection and a target for medical imaging tracing, the preparation of anti-CD318 monoclonal antibodies has broad application potential.

[0005] At present, there are very few antibodies on the market that can specifically and sensitively detect CD318, especially anti-CD318 monoclonal antibodies that can detect CD318 protein on the cell membrane. Therefore, the development and preparation of anti-CD318 monoclonal antibodies with strong specificity, high sensitivity and wide application range has important practical significance and application value. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a hybridoma cell line secreting anti-CD318 monoclonal antibodies and its application, wherein the anti-CD318 monoclonal antibodies secreted by the hybridoma cells have high affinity with CD318 protein, can specifically recognize CD318 protein, and have important application prospects in the preparation of CD318 detection kits. The CD318 detection kit constructed with anti-CD318 monoclonal antibodies has high sensitivity and good specificity, and has good application effects in a variety of detection methods including enzyme-linked immunosorbent assay, flow cytometry and immunofluorescence.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides a hybridoma cell strain 13-8-G2, whose deposit number is CCTCC NO: C202499.

[0009] The present invention also provides the use of the hybridoma cell line 13-8-G2 in preparing anti-CD318 monoclonal antibodies.

[0010] The present invention also provides an anti-CD318 monoclonal antibody, comprising a light chain variable region and a heavy chain variable region;

[0011] The light chain variable region includes CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 6 to 8;

[0012] The heavy chain variable region includes CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 11-13.

[0013] In some specific embodiments of the present invention, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 5; and the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 10.

[0014] In some specific embodiments of the present invention, the anti-CD318 monoclonal antibody is secreted and produced by the hybridoma cell line 13-8-G2 or its successor cell line.

[0015] In some specific embodiments of the present invention, the anti-CD318 monoclonal antibody subtype is IgG1.

[0016] The present invention also provides a nucleic acid molecule encoding the anti-CD318 monoclonal antibody.

[0017] In some specific embodiments of the present invention, the nucleic acid molecule encoding the light chain variable region has a nucleotide sequence as shown in SEQ ID NO:4; the nucleic acid molecule encoding the heavy chain variable region has a nucleotide sequence as shown in SEQ ID NO:9.

[0018] The present invention also provides a method for preparing the anti-CD318 monoclonal antibody, comprising: immunizing mice with an incomplete adjuvant for 1 to 2 weeks, and then intraperitoneally injecting 1 to 2×10 6 The hybridoma cell line 13-8-G2 with a deposit number of CCTCC NO: C202499 was prepared, and the mouse ascites was collected 10 to 14 days later, and the anti-CD318 monoclonal antibody was obtained by purification.

[0019] In some specific embodiments of the invention, the mouse comprises a BALB / c mouse.

[0020] The present invention also provides the use of the anti-CD318 monoclonal antibody in any of the following items:

[0021] (I) preparing an immunoassay product for detecting CD318 protein; and / or

[0022] (II), preparing a product for localizing CD318 protein; and / or

[0023] (III) preparing products for labeling tumor cells.

[0024] In some specific embodiments of the present invention, the product includes a kit, a reagent or a chip.

[0025] The present invention also provides a reagent or a reagent combination, comprising the anti-CD318 monoclonal antibody.

[0026] The present invention also provides a kit, comprising the anti-CD318 monoclonal antibody.

[0027] The present invention also provides a chip, comprising the anti-CD318 monoclonal antibody.

[0028] In some specific embodiments of the present invention, the kit includes a CD318 enzyme-linked immunosorbent assay kit, a CD318 immunofluorescence assay kit, and a CD318 flow cytometry assay kit.

[0029] In some specific embodiments of the present invention, the CD318 enzyme-linked immunosorbent assay kit further comprises: any one or a combination of at least two of a blocking solution, an enzyme-labeled secondary antibody, a washing solution, a color developing solution or a stop solution.

[0030] In some specific embodiments of the present invention, the anti-CD318 monoclonal antibody is modified with a fluorescent group, and the fluorescent group is any one of FITC, Cy3, Cy5, and Alexa Fluor 488.

[0031] In some specific embodiments of the present invention, the anti-CD318 monoclonal antibody in the CD318 flow cytometry detection kit can bind to the CD318 protein on the cell membrane. In the flow cytometry detection, the anti-CD318 monoclonal antibody is used as a primary antibody to detect cells expressing the CD318 protein.

[0032] The present invention provides the following beneficial effects:

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The hybridoma cell line 13-8-G2 provided by the present invention has good passage ability and expansion ability, can stably secrete anti-CD318 monoclonal antibodies, and this secretion ability does not decrease when the culture number increases, and its ability to induce ascites does not decrease with the increase in the number of passages.

[0035] (2) The anti-CD318 monoclonal antibody provided by the present invention has strong specificity, high sensitivity and wide application range. The anti-CD318 monoclonal antibody has a high binding ability with the CD318 protein. Under the same dilution conditions, the anti-CD318 monoclonal antibody secreted by the hybridoma cell line 13-8-G2 has a higher positive OD value for the CD318 protein reaction, indicating that its potency is higher than that of the commercial antibody.

[0036] (3) The anti-CD318 monoclonal antibody provided by the present invention is an IgG1 subtype antibody, which can specifically recognize the CD318 protein on the cell membrane in cell immunofluorescence detection and flow cytometry detection, and has a high affinity.

[0037] Biological Deposit Description

[0038] Biological materials: Hybridoma cell line 13-8-G2 was deposited in China Center for Type Culture Collection on April 2, 2024, at Wuhan University, Wuhan, China, with the deposit number CCTCCNO: C202499. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0040] Figure 1 The plasmid map of pLVX-CD318-IRES-mCherry in Preparation Example 1 of the present invention is shown;

[0041] Figure 2 Shows the fluorescence image of Hela cells infected with pLVX-CD318-IRES-mCherry;

[0042] Figure 3 Figure 2 shows the expression levels of CD318 mRNA and protein in Hela cells and Hela-CD318 cells detected by RT-qPCR (A) and Western Blot (B); 1: Hela cells; 2: Hela-CD318 cells.

[0043] Figure 4 The Coomassie brilliant blue staining image of CD318-mFc recombinant protein purified by Ni column affinity is shown; where M is the protein molecular weight standard; 1: cell supernatant; 2: loading flow-through; 3: 10 mM imidazole washing solution; 4: 20 mM imidazole washing solution; 5: 250 mM imidazole elution solution;

[0044] Figure 5 The figure shows the results of indirect ELISA between the antibodies produced by all hybridoma cell lines secreting anti-CD318 monoclonal antibodies and the eukaryotic expressed human CD318 recombinant protein;

[0045] Figure 6 Figure 2 shows the SDS-PAGE electrophoresis detection diagram of the purified ascites anti-CD318 monoclonal antibody; wherein, M is the protein molecular weight standard; 1 and 2: anti-CD318 monoclonal antibody in the reduced state, 2 μg;

[0046] Figure 7 The subtype identification results of the anti-CD318 monoclonal antibody secreted by the hybridoma cell line 13-8-G2 are shown;

[0047] Figure 8 Shows the sequencing results of the PCR products of the heavy chain and light chain variable regions and the structural analysis of the heavy chain and light chain variable regions;

[0048] Fig. 9 The results of detecting the specificity of anti-CD318 monoclonal antibody by indirect ELISA are shown; wherein, 1: anti-CD318 monoclonal antibody; 2: negative control; 3: positive control antibody 30D4;

[0049] Fig.10The figure shows the result of cell immunofluorescence experiment using CD318 immunofluorescence detection kit;

[0050] Fig.11 Graph showing the results of a cell flow cytometry analysis experiment using a CD318 flow cytometry detection kit. DETAILED DESCRIPTION

[0051] The present invention discloses hybridoma cell line 13-8-G2, anti-CD318 monoclonal antibody and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0052] A hybridoma cell line secreting an anti-CD318 monoclonal antibody is provided. The hybridoma cell line is named hybridoma cell line 13-8-G2, deposited in China Center for Type Culture Collection, with a deposit number of CCTCC NO: C202499, and a deposit date of April 2, 2024.

[0053] Provided is an anti-CD318 monoclonal antibody, wherein the anti-CD318 monoclonal antibody is secreted and produced by the above hybridoma cell line or its passaged cell line.

[0054] In the present invention, the anti-CD318 monoclonal antibody uses human CD318-mFc recombinant protein as an immunogen to immunize mice, and the spleen cells of the immunized mice are fused with the myeloma cells of the mice to form hybridoma cells, and the indirect immunofluorescence method is used to screen the hybridoma cells that can produce highly sensitive and highly specific monoclonal antibodies.

[0055] The method for preparing the anti-CD318 monoclonal antibody of the present invention mainly comprises the following steps:

[0056] (1) Using the eukaryotic expression platform, a eukaryotic expression plasmid was constructed. After sequencing verification, it was transiently expressed in HEK293T cells. Western blot confirmed that the protein could be expressed. The human CD318-mFc recombinant protein was obtained by amplification and transient expression in HEK293F cells and purification.

[0057] (2) BALB / c mice were immunized with human CD318-mFc recombinant protein. After the serum titer was qualified, the mouse spleen cells were fused with mouse myeloma cells SP2 / 0. After multiple screening and subcloning, a hybridoma cell line capable of producing monoclonal antibodies that specifically recognize human CD318 protein was obtained.

[0058] (3) Cultivating hybridoma cell lines, preparing ascites containing anti-CD318 monoclonal antibodies by intraperitoneal inoculation of mice, performing affinity purification to obtain anti-CD318 monoclonal antibodies, and determining the activity of anti-CD318 monoclonal antibodies by ELISA.

[0059] The invention provides a CD318 enzyme-linked immunosorbent assay kit, which comprises an anti-CD318 monoclonal antibody secreted by a hybridoma cell line 13-8-G2.

[0060] Preferably, the CD318 enzyme-linked immunosorbent assay kit further comprises: any one or a combination of at least two of a blocking solution, an enzyme-labeled secondary antibody, a washing solution, a color developing solution or a stop solution.

[0061] The invention provides a CD318 immunofluorescence detection kit, which contains an anti-CD318 monoclonal antibody secreted by a hybridoma cell line 13-8-G2.

[0062] Preferably, the anti-CD318 monoclonal antibody is modified with a fluorescent group, and the fluorescent group is any one of FITC, Cy3, Cy5, and Alexa Fluor 488.

[0063] The present invention provides a CD318 flow cytometry detection kit, which comprises an anti-CD318 monoclonal antibody secreted by a hybridoma cell line 13-8-G2.

[0064] In the invention, the anti-CD318 monoclonal antibody in the CD318 flow cytometry detection kit can bind to the CD318 protein on the cell membrane. In the flow cytometry detection, the anti-CD318 monoclonal antibody is used as a primary antibody to detect cells expressing the CD318 protein.

[0065] Unless otherwise specified, the hybridoma cell line 13-8-G2, anti-CD318 monoclonal antibody and raw materials and reagents used in their application provided by the present invention can all be purchased from the market.

[0066] The present invention will be further described below in conjunction with embodiments:

[0067] Preparation Example 1 Preparation of Hela cell stably transfected cell line overexpressing human CD318

[0068] The preparation process of human CD318 overexpressing Hela cell line is as follows:

[0069] Construction of human CD318 overexpression plasmid: The designed CD318 gene sequence was fully synthesized by Beijing Qingke Biotechnology Co., Ltd., and the target gene fragment was obtained by double restriction digestion with XbaI and BamHI, and inserted into the XbaI and BamHI sites of the pLVX-IRES-mCherry vector to obtain the following: Figure 1 The recombinant lentiviral vector pLVX-CD318-IRES-mCherry is shown, wherein the amino acid sequence of CD318 protein is shown as SEQ ID NO.1.

[0070] SEQ ID NO.1:

[0071] MAGLNCGVSIALLGVLLLGAARLPRGAEAFEIALPRESNITVLIKLGTPTLLAKPCYIVISKRHITMLSIKSGERIVFTFSCQSPENHFVIEIQKNIDCMSGPCPFGEVQLQPSTSSLLPTLNRTFIWDVKAHKSIGLELQFSIPRLRQIGPGESCPDGVTHSISGRIDATVVRIGTFCSNGTVSRIKMQEGVKMALHLPWFHPRNVSGF SIANRSSIKRLCIIESVFEGEGSATLMSANYPEGFPEDELMTWQFVVPAHLRASVSFLNFNLSNCERKEERVEYYIPGSTTNPEVFKLEDKQPGNMAGNFNLSL QGCDQDAQSPGILRLQFQVLVQHPQNESNKIYVVDLSNERAMSLTIEPRPVKQSRKFVPGCFVCLESRTCSSNLTLTSGSKHKISFLCDDLTRLWMNVEKTISCT DHRYCQRKSYSLQVPSDILHLPVELHDFSWKLLVPKDRLSLVLVPAQKLQQHTHEKPCNTSFSYLVASAIPSQDLYFGSFCPGGSIKQIQVKQNISVTLRTFAP SFQQEASRQGLTVSFIPYFKEEGVFTVTPDTKSKVYLRTPNWDRGLPSLTSVSWNISVPRDQVACLTFFKERSGVVCQTGRAFMIIQEQRTRAEEIFSLDEDVLP KPSFHHHSFWVNISNCSPTSGKQLDLLFSVTLTPRTVDLTVILIAAVGGGVLLLSALGLIICCVKKKKKKTNKGPAVGIYNDNINTEMPRQPKKFQKGRKDNDS HVYAVIEDTMVYGHLLQDSSGSFLQPEVDTYRPFQGTMGVCPPSPPTICSRAPTAKLATEEPPPRSPPESESEPYTFSHPNNGDVSSKDTDIPLLNTQEPMEPAE

[0072] The recombinant lentiviral vector overexpressing CD318 was combined with the virus packaging auxiliary plasmids pMD2G and pSPAX2 to co-transfect 293T cells to obtain the virus supernatant; after ultra-fast concentration and purification, the virus supernatant was infected with Hela cells, and the virus infection efficiency was determined under a fluorescence microscope. Figure 2As shown, a CD318 overexpressing Hela cell stable cell line was obtained by limiting dilution method, and the overexpression effect was identified by RT-qPCR and Western Blot. The results are shown in Figure 3 shown.

[0073] Preparation Example 2 Preparation of human CD318-mFc recombinant protein immunogen

[0074] The preparation process of the human CD318-mFc recombinant protein immunogen is as follows:

[0075] mFc and 6 histidines were added to the C-terminus of the extracellular domain of human CD318 protein, and a signal peptide suitable for eukaryotic protein secretion was added to the N-terminus of the protein, wherein mFc refers to the Fc protein fragment of mouse IgG. The target gene was connected to the eukaryotic expression vector pVAX1 by homologous recombination to construct the eukaryotic recombinant plasmid pVAX1-CD318-mFc. The amino acid sequence of the extracellular domain region (Phe30-Leu666) of human CD318 protein and the complete sequence with mFc and 6 histidines added are shown in SEQ ID NO.2.

[0076] SEQ ID NO.2:

[0077] FEIALPRESNITVLIKLGTPTLLAKPCYIVISKRHITMLSIKSGERIVFTFSCQSPENHFVIEIQKNIDCMSGPCPFGEVQLQPSTSLLPTLNRTFIWDVKAHKSIGLELQFSIPRLRQIGPGESCPDGVTHSISGRIDATVVRIGTFCSNGTVSRIKMQEGVKMALHLPWFHPRNVSGFSIANRSSIKRLCIIESVFEGEGSATLMSANYPEGFPEDELMTWQFVVPAHLRASVSFLNFNLSNCERKEERVEYYIPGSTTNPEVFKLEDKQPGNMAGNFNLSLQGCDQDAQSPGILRLQFQVLVQHPQNESNKIYVVDLSNERAMSLTIEPRPVKQSRKFVPGCFVCLESRTCSSNLTLTSGSKHKISFLCDDLTRLWMNVEKTISCTDHRYCQRKSYSLQVPSDILHLPVELHDFSWKLLVPKDRLSLVLVPAQKLQQHTHEKPCNTSFSYLVASAIPSQDLYFGSFCPGGSIKQIQVKQNISVTLRTFAPSFQQEASRQGLTVSFIPYFKEEGVFTVTPDTKSKVYLRTPNWDRGLPSLTSVSWNISVPRDQVACLTFFKERSGVVCQTGRAFMIIQEQRTRAEEIFSLDEDVLPKPSFHHHSFWVNISNCSPTSGKQLDLLFSVTLTPRTVDLPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKHHHHHH

[0078] The constructed eukaryotic expression plasmid pVAX1-CD318-mFc was transiently expressed in 293T cells. After 72 hours, the cell expression supernatant was collected and verified by Western blot to confirm that the protein was expressed. Then the culture was expanded, the plasmid was extracted, and the plasmid DNA was transiently transfected into HEK293F cells. Protein expression was performed in HEK293F cells. Samples were counted at different time points, the cell state was observed, the cell viability was detected, and the expression of the target protein was detected by Western blot. The cell culture was harvested at 96 hours, centrifuged at 10000 rpm for 30 minutes, and the liquid was filtered with a 0.45 μm disposable sterile filter. The supernatant was collected for purification to obtain human CD318-mFc recombinant protein.

[0079] A Ni column was used for affinity purification, and the purified protein was subjected to SDS-PAGE gel electrophoresis to detect its protein size and purity. The results are as follows Figure 4 As shown, the size of human CD318-mFc recombinant protein is about 130 kDa, consistent with the expected band position, and the purity is greater than 95%.

[0080] Preparation Example 3 Preparation of human CD318 recombinant protein

[0081] The preparation process of the human CD318 recombinant protein is as follows:

[0082] Six histidines were added to the C-terminus of the extracellular domain of human CD318 protein, and a signal peptide suitable for eukaryotic protein secretion was added to the N-terminus of the protein. The target gene was connected to the eukaryotic expression vector pVAX1 by homologous recombination to construct the eukaryotic recombinant plasmid pVAX1-CD318. The amino acid sequence of the extracellular domain region (Phe30-Leu666) of human CD318 protein and the complete sequence with six histidines added are shown in SEQ ID NO.3.

[0083] SEQ ID NO.3:

[0084] FEIALPRESNITVLIKLGTPTLLAKPCYIVISKRHITMLSIKSGERIVFTFSCQSPENHFVIEIQKNIDCMSGPCPFGEVQLQPSTSLLPTLNRTFIWDVKAHKSIGLELQFSIPRLRQIGPGESCPDGVTHSISGRIDATVVRIGTFCSNGTVSRIKMQ EGVKMALHLPWFHPRNVSGFSIANRSSIKRLCIIESVFEGEGSATLMSANYPEGFPEDELMTWQFVVPAHLRASVSFLNFNLSNCERKEERVEYYIPGSTTNPEVFKLEDKQPGNMAGNFNLSLQGCDQDAQSPGILRLQFQVLVQHPQNESNKIYVVDLS NERAMSLTIEPRPVKQSRKFVPGCFVCLESRTCSSNLTLTSGSKHKISFLCDDLTRLWMNVEKTISCTDHRYCQRKSYSLQVPSDILHLPVELHDFSWKLLVPKDRLSLVLVPAQKLQQHTHEKPCNTSFSYLVASAIPSQDLYFGSFCPGGSIKQIQVKQ NISVVTLRTFAPSFQQEASRQGLTVSFIPYFKEEGVFTVTPDTKSKVYLRTPNWDRGLPSLTSVSWNISVPRDQVACLTFFKERSGVVCQTGRAFMIIQEQRTRAEEIFSLDEDVLPKPSFHHHSFWVNISNCSPTSGKQLDLLFSVTLTPRTVDLHHHHHH

[0085] The constructed eukaryotic expression plasmid pVAX1-CD318 was transiently expressed in 293T cells. After 72 hours, the cell expression supernatant was collected and verified by Western blot to confirm the protein expression. Then the culture was expanded, the plasmid was extracted, and the plasmid DNA was transiently transfected into HEK293F cells. Protein expression was performed in HEK293F cells. Samples were counted at different time points, the cell state was observed, the cell viability was detected, and the expression of the target protein was detected by Western blot. The cell culture was harvested at 96 hours, centrifuged at 10000 rpm for 30 minutes, and the liquid was filtered with a 0.45 μm disposable sterile filter. The supernatant was collected and affinity purified using a Ni column to obtain human CD318 recombinant protein.

[0086] Preparation Example 4 Preparation of hybridoma cell lines

[0087] The method for preparing a hybridoma cell line comprises the following steps:

[0088] (1) Immunization of mice

[0089] Prepare 3 6-week-old female Balb / c mice, collect blood from the tail vein, and centrifuge to obtain serum as a negative control. The human CD318-mFc recombinant protein purified in Preparation Example 2 was immunized into mice according to the scheme in Table 1. In general, 100 μg of purified human CD318-mFc recombinant protein emulsified with Freund's complete adjuvant was used for the first immunization of mice by intraperitoneal injection. After the first immunization, each mouse received three more injections of 100 μg of purified human CD318-mFc recombinant protein emulsified with Freund's incomplete adjuvant by intraperitoneal and subcutaneous multi-point injection at intervals of 2 weeks. On the 3rd day after the fourth immunization, blood was collected from the tail vein, and serum was obtained by centrifugation. The serum antibody titer was detected by ELISA, and mice with higher titers were selected for shock immunization, intraperitoneal injection, and fusion was performed 3 days later.

[0090] Table 1 Animal immunization scheme

[0091]

[0092] (2) Cell fusion

[0093] Mouse spleen cells with higher titer were mixed with mouse myeloma cells SP2 / 0 at a ratio of 5:1, where mouse spleen cells were about 1×10 8 and SP2 / 0 cells are about 2×10 7 , centrifuged at 1000 rpm for 5 min, and discarded the culture medium; washed once with serum-free DMEM culture medium, centrifuged at 1000 rpm for 5 min, and discarded the culture medium; fused using 50% PEG as a fusion agent, placed the centrifuge tube containing cells in a 37°C water bath, slowly added 1 mL of 50% PEG solution while stirring the cells, let it stand for 1 min, slowly added 10 mL of serum-free DMEM culture medium, centrifuged at 1000 rpm for 5 min, and discarded the supernatant. Add DMEM culture medium containing 20% ​​fetal bovine serum and 1×HAT, and inoculate the cells into a 96-well cell culture plate, 200 μL per well, and culture in a 37°C, 5% CO2 cell culture incubator.

[0094] (3) Screening of hybridoma cells

[0095] The fused cells were cultured in an incubator for 10 to 14 days, with half the medium replaced every 5 days. When the fused cells grew to cover 1 / 3 of the bottom well area, the culture supernatant was taken and the hybridoma cell lines secreting anti-CD318 antibodies were screened by indirect immunofluorescence method. The operation steps of the indirect immunofluorescence method are as follows: the CD318 overexpression Hela cell stable transfection cell line (CD318 high expression) and Hela cells (CD318 negative) obtained in Preparation Example 1 are inoculated into 96-well cell culture plates, 100 μL of cell suspension per well, and cultured in a 37°C, 5% CO2 cell culture incubator overnight; the supernatant is discarded, and each well is rinsed with PBS solution 3 times; the PBS solution is discarded, and the cells are fixed with PBS solution containing 4% paraformaldehyde, and incubated at room temperature (20°C ± 5°C) for 15 min; the fixative is discarded, and each well is rinsed with PBS solution 3 times; the PBS solution is discarded, and a PBS solution containing 1% BSA is added to block for 30 min; the blocking solution is discarded, and the supernatant of the fused cell culture is added as the primary antibody, 50 μL per well, and incubated at room temperature (20°C ± 5°C) for 1 hour; the supernatant is discarded, and each well is rinsed with PBS solution 3 times, each time for 5 min; the secondary antibody (Alexa Fluor® 488-labeled goat anti-mouse IgG (H+L)), incubate at room temperature (20℃±5℃) in the dark for 1 hour; discard the secondary antibody, add PBS solution to each well for 3 times, 5 min each time; observe directly under an inverted fluorescence microscope, green fluorescence appears in the CD318 overexpressing Hela cell stably transfected cell line incubated with hybridoma cell supernatant, and no green fluorescence is observed in Hela cells, which are determined to be positive clones that can be subcloned.

[0096] (4) Construction of hybridoma cell lines

[0097] The positive hybridoma cells were subcloned and sorted by limiting dilution method. After three subcloning and indirect immunofluorescence experiment screening, 13 hybridoma cell lines that can stably secrete anti-human CD318 monoclonal antibodies were obtained. The clone numbers were 13-1-D5, 13-1-F4, 13-2-G2, 13-3-B3, 13-3-D2, 13-3-E6, 13-4-G9, 13-5-B10, 13-5-G6, 13-6-D1, 13-8-G2, 13-10-G7, and 17-1-B1. The antibodies produced by them were then detected and compared by indirect ELISA method. The results are as follows Figure 5Under the same experimental conditions, 6 of the antibodies had the best titers, which were produced by hybridoma cell line 13-2-G2, hybridoma cell line 13-3-B3, hybridoma cell line 13-3-E6, hybridoma cell line 13-5-B10, hybridoma cell line 13-5-G6, and hybridoma cell line 13-8-G2. The antibody clone numbers were 13-2-G2, 13-3-B3, 13-3-E6, 13-5-B10, 13-5-G6, and 13-8-G2. Figure 5 N is a negative control, P is a positive control, and combined with the results of cell immunofluorescence experiment and cell flow cytometry, the clone number of the hybridoma cell line with the best titer, the strongest specificity and the widest application is 13-8-G2.

[0098] The operating steps of the indirect ELISA method are as follows: the ELISA plate was coated with 1 μg / mL human CD318 recombinant protein, 100 μL / well, overnight at 4°C; the coating solution was discarded the next day, washed three times with PBST (pH 7.4, containing 0.1% Tween-20, 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, 0.01 M potassium dihydrogen phosphate), 5% BSA 150 μL was added to each well, and blocked for 2 hours at 37°C; after discarding the blocking solution, the plate was washed three times with PBST, and then incubated with the primary antibody, 100 μL of the diluted antibody solution was added to each well, and the antibody produced by 12 positive hybridoma cells was diluted with 1% BSA to a concentration of 0.5 μg / mL, and the mouse serum with antibody titer after immunization was used as the positive control P, and the non-immunized mouse serum was used as the negative control N, incubated at 37°C for 1 hour, and washed three times with PBST; 100 μL of the diluted antibody solution was added to each well, and the antibody produced by 12 positive hybridoma cells was diluted with 1% BSA to a concentration of 0.5 μg / mL, and the mouse serum with antibody titer after immunization was used as the positive control P, and the mouse serum without immunization was used as the negative control N, and incubated for 1 hour at 37°C, and washed three times with PBST; 100 μL of the diluted antibody solution was added to each well, and the antibody produced by 12 positive hybridoma cells was diluted with 1% BSA to a concentration of 0.5 μg / mL, and the mouse serum with antibody titer after immunization was used as the positive control P, and the mouse serum without immunization was used as the negative control N, and the plate was incubated for μL corresponding to a 1:5000 dilution (H+L), incubate with secondary antibody and then wash, incubate at 37℃ for 1 hour, wash 3 times with PBST; add 100 μL TMB colorimetric solution to each well, develop at 37℃ for 10 min, add stop solution, and measure the absorbance at a wavelength of 450 nm with an enzyme-labeled instrument.

[0099] (5) Deposit of hybridoma cell line 13-8-G2

[0100] The hybridoma cell with clone number 13-8-G2 was preserved and named hybridoma cell line 13-8-G2, and deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: C202499 and the deposit date of April 2, 2024.

[0101] Preparation Example 5 Preparation of anti-CD318 monoclonal antibody

[0102] The anti-CD318 monoclonal antibody was produced by the hybridoma cell line 13-8-G2 described in Preparation Example 4.

[0103] Three 10-week-old female Balb / c mice were prepared and intraperitoneally injected with 500 μL of Freund's incomplete adjuvant per mouse. One week later, hybridoma cell line 13-8-G2 was intraperitoneally injected with 1-2×10 6 Cells / mouse. After 10-14 days, the mouse ascites was collected according to the swelling of the mouse abdomen. The collected ascites was centrifuged at 4℃ 12000 rpm for 10 min, and the yellow clear liquid in the middle was collected. The antibody was purified by Protein A / G affinity chromatography, the antibody concentration was determined by Nanodrop, and the purity was identified by SDS-PAGE gel electrophoresis. SDS-PAGE gel electrophoresis Figure 6 As shown in the figure, the purity of the purified anti-CD318 monoclonal antibody is >95%, and IgG heavy chain with a molecular weight of 50 kDa and light chain with a molecular weight of 25 kDa can be seen. M is the protein molecular weight standard; 1 and 2: anti-CD318 monoclonal antibody in a reduced state, 2 μg.

[0104] Preparation Example 6 Preparation of CD318 ELISA kit

[0105] The CD318 ELISA kit includes the anti-CD318 monoclonal antibody obtained in Preparation Example 5, a blocking solution and an enzyme-labeled secondary antibody, a washing solution, a TMB colorimetric solution, and a stop solution. The blocking solution is 5% BSA; the enzyme-labeled secondary antibody is (H+L) and (H+L); the washing solution is PBST buffer (pH 7.4, containing 0.1% Tween-20, 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, 0.01 M potassium dihydrogen phosphate); the stop solution is 2 mol / L H2SO4.

[0106] Preparation Example 7 Preparation of CD318 Immunofluorescence Detection Kit

[0107] The CD318 immunofluorescence detection kit includes a fluorescent group-modified anti-CD318 monoclonal antibody, a cell fixative, a blocking solution, a washing solution, and a cell nucleus dye, wherein the fluorescent group is any one of FITC, Cy3, Cy5, and Alexa Fluor 488. The operation process of fluorescent group-modified anti-CD318 monoclonal antibody is described by taking FITC labeling as an example: FITC solution is added dropwise to the anti-CD318 monoclonal antibody solution obtained in Preparation Example 5 under magnetic stirring, and then stirred continuously at room temperature (20°C ± 5°C) for 4 to 6 hours, and then centrifuged. The supernatant is the FITC-labeled anti-CD318 monoclonal antibody, and the labeled antibody is further purified by dialysis to remove unbound free fluorescein.

[0108] Preparation Example 8 Preparation of CD318 Flow Cytometry Detection Kit

[0109] The CD318 flow cytometry detection kit comprises the anti-CD318 monoclonal antibody obtained in Preparation Example 5, an antibody diluent, a cell fixative, a washing solution and a fluorescent secondary antibody, wherein the fluorescent secondary antibody is .

[0110] Example 1 Identification of subtypes of anti-CD318 monoclonal antibodies

[0111] In this example, the subtype of the anti-CD318 monoclonal antibody secreted by the hybridoma cell line 13-8-G2 described in Preparation Example 4 was identified by ELISA.

[0112] The specific steps are as follows:

[0113] The anti-CD318 monoclonal antibody secreted by hybridoma cell line 13-8-G2 was detected using the mouse monoclonal antibody subtype identification kit of proteintech. The specific process is as follows: the supernatant of hybridoma cell line 13-8-G2 was diluted 1:50 and 50 μL was added to the sample wells of the ELISA plate strips, and 50 μL of HRP-labeled secondary antibodies of each subtype antibody were added to each well. After gently mixing on a mixer, incubate at room temperature (20℃±5℃) for 1 hour, discard the liquid in the well, wash the plate 3 times with 1×PBST, pat dry on absorbent paper, add the prepared color development solution to the well, color at room temperature (20℃±5℃) in the dark for 10~20 minutes, then add the stop solution to each well, and read the OD450 by an enzyme reader. Figure 7 The subtype identification result of the anti-CD318 monoclonal antibody secreted by the hybridoma cell line 13-8-G2 provided in this example shows that the anti-CD318 monoclonal antibody is IgG1, and the light chain is kappa type.

[0114] Example 2 Amplification and analysis of the variable region gene of the anti-CD318 monoclonal antibody

[0115] First, the total RNA was extracted from the hybridoma cell line 13-8-G2 described in Preparation Example 4 using the Trizol method: 1×10 6 10 positive hybridoma cells, discard the supernatant, wash once with PBS buffer, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 mL Trizol reagent to the cell pellet, mix repeatedly with a pipette and transfer to a 1.5 mL EP tube, and leave at room temperature for 5 min to ensure that the cells are fully lysed. Add 200 μL chloroform, shake vigorously to mix thoroughly, place at room temperature for 5 min, centrifuge at 4 ℃, 12000 rpm for 15 min, aspirate about 300 μL of the upper liquid and transfer it to a new 1.5 mL EP tube, add 0.5 mL isopropanol, gently invert several times to mix, place at room temperature for 10 min, then centrifuge at 4 ℃, 12000 rpm for 10 min, gelatinous RNA precipitation can be seen at the bottom of the tube, carefully aspirate the supernatant, add 1 mL 75% ethanol, gently invert the centrifuge tube to suspend the precipitation, then centrifuge at 4 ℃, 12000 rpm for 5 min, discard the supernatant, dry the EP tube at room temperature for 5 min to fully evaporate the ethanol, add RNase-free double distilled water to dissolve the precipitation, and obtain total RNA. Reverse transcription kit was used to synthesize cDNA with the extracted total RNA as a template.

[0116] Degenerate primers were designed to amplify the light chain variable region and heavy chain variable region of monoclonal antibody 13-8-G2. The sequencing results of the heavy chain and light chain variable region PCR products and the structural analysis of the heavy chain and light chain variable regions are shown in Figure 2. Figure 8 The obtained monoclonal antibody 13-8-G2 light chain variable region has a size of 318 bp, a nucleotide sequence as shown in SEQ ID NO:4, and an amino acid sequence as shown in SEQ ID NO:5, including CDR1 as shown in SEQ ID NO:6, CDR1 as shown in SEQ ID NO:7, and CDR3 as shown in SEQ ID NO:8. The monoclonal antibody 13-8-G2 heavy chain variable region has a size of 357 bp, a nucleotide sequence as shown in SEQ ID NO:9, and an amino acid sequence as shown in SEQ ID NO:10, including CDR1 as shown in SEQ ID NO:11, CDR2 as shown in SEQ ID NO:12, and CDR3 as shown in SEQ ID NO:13.

[0117] SEQ ID NO:4

[0118] GACATTGTGCTCACCCAGTCTCCAGCAGTCATGTCTGCATTTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCTCCTCAAGTGTAACTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAGCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCACCAACATGGAGGCTGAAGATGCTGCCACTTATTTCTGCCAGCAGTGGACTGGTAACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0119] SEQ ID NO:5

[0120] DIVLTQSPAVMSAFPGEKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTITNMEAEDAATYFCQQWTGNPLTFGAGTKLELK

[0121] SEQ ID NO:6

[0122] SASSSVTYMH

[0123] SEQ ID NO:7

[0124] DTSKLAS

[0125] SEQ ID NO:8

[0126] QQWTGNPLT

[0127] SEQ ID NO:9

[0128] GAGGTGCAGCTGCAGGAGTCTGGACCTAGCCTCGTGAAACCTTCTCAGACTCTGTCCCTCACTTGTTCTGTCACTGGCGACTCCATCACCAGTGGTTACTGGAACTGGATCCGGAAATTCCCTGGGCATAAACTTGAATACTTGGGGTCCATAAGTTACAGTGGTTACACTTTCTACA ATCCATCTCTCAAAAGTCAATTCTCCATCTCTCGAGACACATCCAAGAACCAGTACTACCTGCAGTTGAATTCTGTGACTTCTGAGGACACAGCCACATATTACTGTGCAAGATCGGATGGTAACTACGACTATTCTATGGACTACTGGGGTCACGGAACCTCAGTCACCGTCTCCTCA

[0129] SEQ ID NO:10

[0130] EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGHKLEYLGSISYSGYTFYNPSLKSQFSISRDTSKNQYYLQLNSVTSEDTATYYCARSDGNYDYSMDYWGHGTSVTVSS

[0131] SEQ ID NO:11

[0132] SGWY

[0133] SEQ ID NO:12

[0134] SISYSGYTFYNPSLKS

[0135] SEQ ID NO:13

[0136] SDGNYDYSMDY

[0137] Example 3 Detection of human CD318 recombinant protein using CD318 ELISA kit

[0138] This example uses the CD318 ELISA kit described in Preparation Example 6 to detect the sample:

[0139] The purified human CD318 recombinant protein in Preparation Example 3 was used to coat the ELISA plate, and the specificity of the anti-CD318 monoclonal antibody secreted by the hybridoma cell line 13-8-G2 was detected by indirect ELISA. The positive control antibody was the recombinant anti-CDCP1 antibody (Human IgG) of Invitrogen, clone number 30D4 (Cat. No.: MA5-50097).

[0140] The specific operation steps are as follows: coat the ELISA plate with 1 μg / mL human CD318 recombinant protein, 100 μL / well, overnight at 4°C; discard the coating solution the next day, wash three times with PBST, add 150 μL of 5% BSA to each well, and block at 37°C for 2 hours; discard the blocking solution, wash three times with PBST, and then incubate with the primary antibody, dilute the anti-CD318 monoclonal antibody and the control antibody 30D4 with 1% BSA to a concentration of 0.5 μg / mL, and the negative control is 1% BSA, add 100 μL to each well, incubate at 37°C for 1 hour, and wash three times with PBST; add 100 μL of the corresponding 1:5000 diluted (H+L) and (H+L), incubate with secondary antibody and then wash, incubate at 37℃ for 1 hour, wash 3 times with PBST; add 100 μL TMB colorimetric solution to each well, develop at 37℃ for 10 min, add stop solution, and measure the absorbance at a wavelength of 450 nm with an enzyme reader.

[0141] The results of the ELISA experiment are as follows Fig. 9 As shown, 1: anti-CD318 monoclonal antibody; 2: negative control; 3: positive control antibody 30D4, the anti-CD318 monoclonal antibody secreted by the hybridoma cell line 13-8-G2 and the positive control antibody 30D4 can specifically recognize human CD318 recombinant protein, and the results are both strongly positive.

[0142] Example 4 Determination of affinity constants of anti-CD318 monoclonal antibodies

[0143] In this example, the affinity constant of the anti-CD318 monoclonal antibody described in Preparation Example 5 was determined.

[0144] The ELISA plate was coated with 1 μg / mL human CD318 recombinant protein obtained in Preparation Example 3, 100 μL / well, overnight at 4°C; the next day, the coating solution was discarded, washed 3 times with PBST, 150 μL of 5% BSA was added to each well, and blocked at 37°C for 2 hours; after discarding the blocking solution, the plate was washed 3 times with PBST, and then incubated with the primary antibody, and the purified monoclonal antibody in Preparation Example 5 (initial concentration of 0.5 mg / mL) was diluted 2-fold from 1:200, for a total of 13 gradients, 1 well was left as a blank control, 100 μL was added to each well, incubated at 37°C for 1 hour, and washed 3 times with PBST; (H+L) was diluted 1:5000, 100 μL was added to each well, incubated at 37°C for 1 hour, and washed 3 times with PBST; 100 μL of TMB colorimetric solution was added to each well, color developed at 37°C for 10 min, and after adding the stop solution, the absorbance value at a wavelength of 450 nm was measured with an enzyme marker. The test results are shown in Table 2. The dilution factor A corresponding to half of the maximum binding OD value was found, and the affinity constant was calculated using the following formula: affinity constant = (150000×A) / original antibody concentration.

[0145] Table 2 Affinity constant determination of anti-CD318 monoclonal antibodies

[0146]

[0147] The affinity constant of the anti-CD318 monoclonal antibody of the present invention is calculated by the above formula to be approximately 7.68×10 9 The present invention provides an anti-CD318 monoclonal antibody having a high affinity to the CD318 protein.

[0148] Example 5 Detection of intracellular CD318 using CD318 immunofluorescence detection kit

[0149] In this example, the CD318 immunofluorescence detection kit described in Preparation Example 7 was used to detect various cells.

[0150] Hela cells, Hela-CD318 cells, A549 cells, NCI-H460 cells, HT-29 cells and HCT116 cells were inoculated in 24-well plates with cell slides, and cultured in an incubator overnight; washed three times with PBS buffer (pH 7.4, containing 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, 0.01 M potassium dihydrogen phosphate), fixed with 4% paraformaldehyde for 15 min, washed three times with PBS buffer, and blocked with 5% BSA solution at 37°C for 2 hours; after discarding the blocking solution, washed three times with PBS buffer, and then incubated with primary antibody, and incubated with 1% Dilute FITC-labeled anti-CD318 monoclonal antibody with BSA, incubate overnight at 4°C in the dark, wash three times with PBS buffer, add DAPI solution to stain the cell nucleus, wash three times with PBS buffer, dry the liquid on the slide with absorbent paper, seal the slide with sealing solution containing anti-fluorescence quencher, observe and take pictures under a fluorescence microscope, and the results are as follows: Fig.10 As shown. Fig.10 It can be seen that the anti-CD318 monoclonal antibody provided by the present invention can specifically recognize CD318 protein.

[0151] Example 6 Detection of intracellular CD318 using a flow cytometry detection kit

[0152] In this example, the flow cytometry detection kit described in Preparation Example 8 was used to detect a variety of cells.

[0153] Hela cells, Hela-CD318 cells, A549 cells, NCI-H460 cells, HT-29 cells and HCT116 cells were treated separately to obtain single cell suspensions, anti-CD318 monoclonal antibodies diluted in PBS buffer containing 3% BSA were added, and the suspensions were incubated at room temperature (20℃±5℃) or 4℃ for 30 min, and the cells were washed 3 times with ice-cold PBS buffer. Fluorescent secondary antibodies diluted in PBS buffer containing 3% BSA were added, and the cells were resuspended. The suspensions were incubated at room temperature (20℃±5℃) or 4℃ in the dark for 30 min, and the cells were washed 3 times with ice-cold PBS buffer. The cells were resuspended with PBS buffer and analyzed by flow cytometry. If the cells cannot be put on the machine in time, the cells can be resuspended with 1% paraformaldehyde and stored at 4℃, but this process is not recommended to exceed 16 hours. The test results are as follows: Fig.11 shown.

[0154] Depend on Fig.10It can be seen that the antibody has almost no binding with the membrane surface antigen of Hela cells (CD318 negative), but has good binding with the membrane surface antigen of other CD318 positive cells, and the binding rates with the membrane surface antigen of Hela-CD318 cells, A549 cells, NCI-H460 cells, HT-29 cells and HCT116 cells are 94.3%, 64.2%, 46.0%, 100.0% and 98.5% respectively. The anti-CD318 monoclonal antibody provided by the present invention can specifically recognize CD318 protein, and has important application prospects in the preparation of CD318 detection kits.

[0155] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Hybridoma cell line 13-8-G2, characterized in that: Its deposit number is CCTCC NO: C202499.

2. Use of the hybridoma cell line 13-8-G2 as claimed in claim 1 in the preparation of anti-CD318 monoclonal antibodies.

3. An anti-CD318 monoclonal antibody, characterized in that including a light chain variable region and a heavy chain variable region; The light chain variable region includes CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 6 to 8; The heavy chain variable region includes CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 11-13.

4. The anti-CD318 monoclonal antibody according to claim 3, characterized in that The anti-CD318 monoclonal antibody is secreted and produced by the hybridoma cell line 13-8-G2 or its successive cell line according to claim 1.

5. A nucleic acid molecule encoding the anti-CD318 monoclonal antibody according to claim 3 or 4.

6. The method for preparing an anti-CD318 monoclonal antibody according to claim 3 or 4, characterized in that: include: After immunizing mice with incomplete adjuvant for 1-2 weeks, 1-2×10 6 The hybridoma cell line 13-8-G2 with a deposit number of CCTCC NO: C202499 was prepared, and the mouse ascites was collected 10 to 14 days later, and the anti-CD318 monoclonal antibody was obtained by purification.

7. Use of the anti-CD318 monoclonal antibody according to claim 3 or 4 in any of the following items: (I) preparing an immunoassay product for detecting CD318 protein; and / or (II), preparing a product for localizing CD318 protein; and / or (III) preparing products for labeling tumor cells.

8. A reagent or a reagent combination, characterized in that Comprising the anti-CD318 monoclonal antibody as described in claim 3 or 4.

9. A kit, characterized in that Comprising the anti-CD318 monoclonal antibody as described in claim 3 or 4.

10. A chip, characterized in that Comprising the anti-CD318 monoclonal antibody as described in claim 3 or 4.

Images