Hybridoma cell line 13-8-g2, anti-cd318 monoclonal antibody and uses thereof

Abstract

The present application relates to the technical field of biotechnology, in particular to hybridoma cell strain 13-8-G2, anti-CD318 monoclonal antibody and application thereof.The light chain variable region of the anti-CD318 monoclonal antibody provided by the present application comprises CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:7 and CDR3 shown in SEQ ID NO:8; the heavy chain variable region comprises CDR1 shown in SEQ ID NO:11, CDR2 shown in SEQ ID NO:12 and CDR3 shown in SEQ ID NO:13, which has high affinity with CD318 protein, can specifically recognize CD318 protein, has higher titer than commercialized antibody, and has important application prospect in preparation of CD318 detection kit.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the hybridoma cell line 13-8-G2, the anti-CD318 monoclonal antibody, and their applications. Background Technology

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

[0003] Numerous studies have shown that CD318 is significantly upregulated in various malignant tumors, such as pancreatic cancer, renal cell carcinoma, liver cancer, breast cancer, lung cancer, colorectal cancer, prostate cancer, and melanoma, while it is almost not expressed or expressed at low levels in normal tissues. High expression of CD318 is associated with tumor development, progression, and poor prognosis. It has been reported that various tumor-associated proteins interact with CD318. CD318 is a key hub in several classic signaling pathways of tumorigenesis 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 range 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 in carcinogenic signaling, CD318 can serve as a tumor biomarker, and its detection can be used as a basis for diagnosing tumor development and treatment efficacy. CD318 can also be used as a target for PET radiotracers for tumor imaging. Furthermore, CD318 and its mediated signaling pathways can also serve as targets for antibody drugs in cancer treatment. Additionally, CD318 can be a target for ADC drugs, and anti-CD318 antibodies can deliver cytotoxic payloads. Currently, several pharmaceutical companies are developing antibody drugs and ADC drugs targeting CD318, and these projects are all in the preclinical stage. The high expression of CD318 in various tumor cells makes it suitable for the development of antibody drugs, ADCs, CAR-T therapies, etc. Combining its applications as a biomarker for tumor development and prognosis, as well as its role as a medical imaging tracer target, the preparation of anti-CD318 monoclonal antibodies has broad application potential.

[0005] Currently, there are very few antibodies on the market that can specifically and sensitively detect CD318, especially anti-CD318 monoclonal antibodies that can detect the CD318 protein on the cell membrane. Therefore, developing and preparing anti-CD318 monoclonal antibodies with strong specificity, high sensitivity, and a wide range of applications has significant practical importance and application value. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a hybridoma cell line that secretes an anti-CD318 monoclonal antibody and its application. The anti-CD318 monoclonal antibody secreted by the hybridoma cells has a high affinity for the CD318 protein and can specifically recognize the CD318 protein, showing significant application potential in the preparation of CD318 detection kits. CD318 detection kits constructed with anti-CD318 monoclonal antibodies exhibit high sensitivity and specificity, and show good performance in various detection methods, including enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunofluorescence.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a hybridoma cell line 13-8-G2, whose accession number is CCTCC NO: C202499.

[0009] The present invention also provides the application of the hybridoma cell line 13-8-G2 in the preparation of anti-CD318 monoclonal antibody.

[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 amino acid sequences as shown in SEQ ID NO: 6-8;

[0012] The heavy chain variable region includes CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO: 11-13.

[0013] In some specific embodiments of the present invention, the light chain variable region includes an amino acid sequence as shown in SEQ ID NO: 5; the heavy chain variable region includes 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 by the hybridoma cell line 13-8-G2 or its passaged 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 aforementioned 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 aforementioned anti-CD318 monoclonal antibody, comprising: immunizing mice with an incomplete adjuvant for 1-2 weeks, and then injecting the mice intraperitoneally with 1-2 × 10⁻⁶ oz. 6 The hybridoma cell line 13-8-G2 with accession number CCTCC NO: C202499 was used. Mouse ascites fluid was collected after 10-14 days, purified, and the anti-CD318 monoclonal antibody was obtained.

[0019] In some specific embodiments of the present invention, the mice include BALB / c mice.

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

[0021] (I) Preparation of immunoassay products for detecting CD318 protein; and / or

[0022] (II) Preparation of products targeting the CD318 protein; and / or

[0023] (III) Preparation of products labeled with tumor cells.

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

[0025] The present invention also provides reagents or combinations of reagents, including the anti-CD318 monoclonal antibody.

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

[0027] The present invention also provides a chip comprising the aforementioned 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 includes any one or at least two combinations of blocking solution, enzyme-labeled secondary antibody, washing solution, colorimetric solution or stop solution.

[0030] In some specific embodiments of the present invention, the anti-CD318 monoclonal antibody is modified with a fluorescent group, wherein 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 flow cytometry detection, the anti-CD318 monoclonal antibody is used as a primary antibody to detect cells expressing the CD318 protein.

[0032] This 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 and expansion capabilities, can stably secrete anti-CD318 monoclonal antibody, and this secretion capability does not decrease with the increase of culture passages, nor does its ability to induce ascites decrease with the increase of passages.

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

[0037] Biological Preservation Instructions

[0038] Biological material: Hybridoma cell line 13-8-G2 was deposited on April 2, 2024, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCCNO: C202499. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0040] Figure 1 This shows the plasmid pattern of pLVX-CD318-IRES-mCherry in Preparation Example 1 of the present invention;

[0041] Figure 2 Fluorescence pattern of HeLa cells infected with pLVX-CD318-IRES-mCherry;

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

[0043] Figure 4 Coomassie Brilliant Blue staining image of CD318-mFc recombinant protein purified by Ni column affinity; where M is the protein molecular weight standard; 1: cell supernatant; 2: sample loading fluid; 3: 10 mM imidazole washing buffer; 4: 20 mM imidazole washing buffer; 5: 250 mM imidazole elution buffer;

[0044] Figure 5 Figure showing the results of indirect ELISA detection of antibodies produced by all hybridoma cell lines secreting anti-CD318 monoclonal antibodies and recombinant human CD318 protein expressed in eukaryotes;

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

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

[0047] Figure 8 Sequencing results of PCR products from the variable regions of the heavy and light chains, and structural analysis of the variable regions of the heavy and light chains;

[0048] Figure 9 The results of detecting the specificity of anti-CD318 monoclonal antibody using indirect ELISA are shown; where 1: anti-CD318 monoclonal antibody; 2: negative control; 3: positive control antibody 30D4;

[0049] Figure 10The results of cell immunofluorescence experiments using the CD318 immunofluorescence detection kit are shown in the figure.

[0050] Figure 11 The figure shows the results of a cell flow cytometry analysis experiment using the CD318 flow cytometry detection kit. Detailed Implementation

[0051] This invention discloses the hybridoma cell line 13-8-G2, the anti-CD318 monoclonal antibody, and their applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this 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 and is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C202499 and deposit date of April 2, 2024.

[0053] A monoclonal antibody against CD318 is provided, wherein the monoclonal antibody against CD318 is secreted by the hybridoma cell line or its passaged cell line described above.

[0054] In this invention, the anti-CD318 monoclonal antibody uses human CD318-mFc recombinant protein as an immunogen to immunize mice. After immunization, the spleen cells of the mice are fused with myeloma cells to form hybridoma cells. Hybridoma cells that can produce highly sensitive and high-specificity monoclonal antibodies are screened using indirect immunofluorescence.

[0055] The preparation method of the anti-CD318 monoclonal antibody described in this invention mainly includes the following steps:

[0056] (1) Using a 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 protein was then expanded and cultured in HEK293F cells, transiently expressed, and purified to obtain recombinant human CD318-mFc protein.

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

[0058] (3) Culture hybridoma cell lines, prepare ascites containing anti-CD318 monoclonal antibody by mouse intraperitoneal inoculation method, perform affinity purification to obtain anti-CD318 monoclonal antibody, and determine the activity of anti-CD318 monoclonal antibody by ELISA.

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

[0060] Preferably, the CD318 enzyme-linked immunosorbent assay kit further includes any one or at least two combinations of blocking solution, enzyme-labeled secondary antibody, washing solution, colorimetric solution or stop solution.

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

[0062] Preferably, the anti-CD318 monoclonal antibody is modified with a fluorescent group, wherein 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, wherein the CD318 flow cytometry detection kit contains an anti-CD318 monoclonal antibody secreted by hybridoma cell line 13-8-G2.

[0064] In this invention, the anti-CD318 monoclonal antibody in the CD318 flow cytometry detection kit can bind to the CD318 protein on the cell membrane. In 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, the anti-CD318 monoclonal antibody, and the raw materials and reagents used in the application of the present invention can all be purchased from the market.

[0066] The present invention will be further illustrated below with reference to the embodiments:

[0067] Preparation Example 1: Preparation of a stable HeLa cell line overexpressing human CD318

[0068] The preparation process of the 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. The target gene fragment was obtained by double digestion with XbaI and BamHI, and then inserted into the XbaI and BamHI sites of the pLVX-IRES-mCherry vector to obtain the desired overexpression plasmid. Figure 1 The recombinant lentiviral vector pLVX-CD318-IRES-mCherry shown here contains the CD318 protein amino acid sequence as shown in SEQ ID NO.1.

[0070] SEQ ID NO.1:

[0071] MAGLNCGVSIALLGVLLLGAARLPRGAEAFEIALPRESNITVLIKLGTPTLLAKPCYIVISKRHITMLSIKSGERIVFTFSCQSPENHFVIEIQKNIDCMSGPCPFGEVQLQPSTSSLLPTLNRTFIWDVKAHKSIGLELQFSIPRLRQIGPGESCPDGVTHSISGRIDATVVRIGTFCSNGTVSRIKMQEGVKMALHLPWFHPRNVSGF SIANRSSIKRLCIIESVFEGEGSATLMSANYPEGFPEDELMTWQFVVPAHLRASVSFLNFNLSNCERKEERVEYYIPGSTTNPEVFKLEDKQPGNMAGNFNLSL QGCDQDAQSPGILRLQFQVLVQHPQNESNKIYVVDLSNERAMSLTIEPRPVKQSRKFVPGCFVCLESRTCSSNLTLTSGSKHKISFLCDDLTRLWMNVEKTISCT DHRYCQRKSYSLQVPSDILHLPVELHDFSWKLLVPKDRLSLVLVPAQKLQQHTHEKPCNTSFSYLVASAIPSQDLYFGSFCPGGSIKQIQVKQNISVTLRTFAP SFQQEASRQGLTVSFIPYFKEEGVFTVTPDTKSKVYLRTPNWDRGLPSLTSVSWNISVPRDQVACLTFFKERSGVVCQTGRAFMIIQEQRTRAEEIFSLDEDVLP KPSFHHHSFWVNISNCSPTSGKQLDLLFSVTLTPRTVDLTVILIAAVGGGVLLLSALGLIICCVKKKKKKTNKGPAVGIYNDNINTEMPRQPKKFQKGRKDNDS HVYAVIEDTMVYGHLLQDSSGSFLQPEVDTYRPFQGTMGVCPPSPPTICSRAPTAKLATEEPPPRSPPESESEPYTFSHPNNGDVSSKDTDIPLLNTQEPMEPAE

[0072] A recombinant lentiviral vector overexpressing CD318 was co-transfected into 293T cells with viral packaging helper plasmids pMD2G and pSPAX2 to obtain viral supernatant. After ultraconcentration and purification, the viral supernatant was used to infect HeLa cells, and the viral infection efficiency was determined under a fluorescence microscope. Figure 2As shown, a stable CD318-overexpressing HeLa cell line was obtained using the limiting dilution method. The overexpression effect was identified by RT-qPCR and Western Blot, and the results are as follows. Figure 3 As 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] An mFc and six histidine residues were added to the C-terminus of the extracellular domain of the human CD318 protein, and a signal peptide suitable for eukaryotic protein secretion was added to the N-terminus. Here, mFc refers to the Fc protein fragment of mouse IgG. The target gene was ligated to the eukaryotic expression vector pVAX1 using homologous recombination to construct the eukaryotic recombinant plasmid pVAX1-CD318-mFc. The amino acid sequence of the extracellular domain region (Phe30-Leu666) of the human CD318 protein and the complete sequence with mFc and six histidine residues 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 transfected into HEK293T cells. After 72 hours, the cell expression supernatant was collected and verified by Western blot, confirming protein expression. The cells were then expanded, the plasmid was extracted, and the plasmid DNA was transiently transfected into HEK293F cells for protein expression. Cell counts were performed at different time points to observe cell status and viability, and the expression of the target protein was detected by Western blot. The cell culture was harvested at 96 hours, centrifuged at 10,000 rpm for 30 min, and filtered through a 0.45 μm disposable sterile filter. The supernatant was collected for purification to obtain the recombinant human CD318-mFc protein.

[0079] Affinity purification was performed using a Ni column. The purified protein was then subjected to SDS-PAGE gel electrophoresis to determine its size and purity. The results are as follows: Figure 4 As shown, the human CD318-mFc recombinant protein is approximately 130 kDa in size, which matches the expected band position, and its purity is greater than 95%.

[0080] Preparation Example 3: Preparation of Recombinant Human CD318 Protein

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

[0082] Six histidine residues were added to the C-terminus of the extracellular domain of the human CD318 protein, and a signal peptide suitable for eukaryotic protein secretion was added to the N-terminus. The target gene was then linked to the eukaryotic expression vector pVAX1 using homologous recombination to construct the eukaryotic recombinant plasmid pVAX1-CD318. The amino acid sequence of the extracellular domain region (Phe30-Leu666) of the human CD318 protein and the complete sequence with the six histidine residues 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 transfected into HEK293T cells. After 72 hours, the cell expression supernatant was collected and verified by Western blot, confirming protein expression. The cells were then expanded, the plasmid was extracted, and the plasmid DNA was transiently transfected into HEK293F cells for protein expression. Cell counts were performed at different time points to observe cell state and viability, and the expression of the target protein was detected by Western blot. The cell culture was harvested at 96 hours, centrifuged at 10,000 rpm for 30 min, filtered through a 0.45 μm disposable sterile filter, and the supernatant was collected. Affinity purification using a Ni column yielded recombinant human CD318 protein.

[0086] Preparation Example 4: Preparation of Hybridoma Cell Lines

[0087] The method for preparing hybridoma cell lines includes the following steps:

[0088] (1) Immunizing mice

[0089] Three 6-week-old female Balb / c mice were used. Blood was collected from the tail vein, and serum was obtained by centrifugation as a negative control. The purified human CD318-mFc recombinant protein obtained in Preparation Example 2 was used to immunize the mice according to the protocol in Table 1. Generally, 100 μg of purified human CD318-mFc recombinant protein emulsified with Freund's complete adjuvant was administered intraperitoneally for the initial immunization. After the initial immunization, each mouse received three more injections of 100 μg of purified human CD318-mFc recombinant protein emulsified with Freund's incomplete adjuvant at two-week intervals via intraperitoneal and subcutaneous multiple-point injections. On day 3 after the fourth immunization, blood was collected from the tail vein, and serum was obtained by centrifugation. Serum antibody titers were detected by ELISA. Mice with higher titers were selected for a pulse immunization via intraperitoneal injection, and fusion was performed 3 days later.

[0090] Table 1 Animal Immunization Program

[0091]

[0092] (2) Cell fusion

[0093] Mouse spleen cells with high potency were mixed with mouse myeloma cells SP2 / 0 at a ratio of 5:1, with approximately 1 × 10⁻⁶ mouse spleen cells. 8 Approximately 2 × 10⁶ SP2 / 0 cells. 7 Centrifuge at 1000 rpm for 5 min and discard the culture medium. Wash once with serum-free DMEM medium, centrifuge at 1000 rpm for 5 min and discard the culture medium. Use 50% PEG as a fusion agent for fusion. Place the centrifuge tube containing cells in a 37°C water bath, and slowly add 1 mL of 50% PEG solution while stirring the cells. After standing for 1 min, slowly add 10 mL of serum-free DMEM medium, centrifuge at 1000 rpm for 5 min and discard the supernatant. Add DMEM medium containing 20% ​​fetal bovine serum and 1×HAT, and seed the cells into 96-well cell culture plates, 200 μL per well, and incubate at 37°C in a 5% CO2 cell culture incubator.

[0094] (3) Screening of hybridoma cells

[0095] The fused cells were cultured in an incubator for 10-14 days, with half of the medium being changed every 5 days. When the fused cells grew to cover 1 / 3 of the bottom well area, the culture supernatant was collected and hybridoma cell lines secreting anti-CD318 antibodies were screened using indirect immunofluorescence. The procedure for indirect immunofluorescence assay is as follows: The CD318-overexpressing HeLa cell line (high CD318 expression) and HeLa cells (CD318 negative) obtained in Example 1 were seeded into 96-well cell culture plates, 100 μL of cell suspension per well, and incubated overnight at 37°C in a 5% CO2 cell culture incubator; the supernatant was discarded, and each well was washed three times with PBS solution; the PBS solution was discarded, and the cells were fixed with PBS solution containing 4% paraformaldehyde, and incubated at room temperature (20°C ± 5°C) for 15 min; the fixative was discarded, and each well was washed three times with PBS solution; the PBS solution was discarded, and the cells were blocked with PBS solution containing 1% BSA for 30 min; the blocking solution was discarded, and 50 μL of the fusion cell culture supernatant was added as primary antibody per well, and incubated at room temperature (20°C ± 5°C) for 1 hour; the supernatant was discarded, and each well was washed three times with PBS solution for 5 min each time; the secondary antibody (Alexa Fluor®) diluted 1:1000 with the blocking solution was added. 488-labeled goat anti-mouse IgG (H+L) was incubated at room temperature (20℃±5℃) in the dark for 1 hour; the secondary antibody was discarded, and each well was rinsed 3 times with PBS solution for 5 min each time; the cells were observed directly under an inverted fluorescence microscope. Green fluorescence appeared in the CD318 overexpressing HeLa cell line incubated with the hybridoma cell supernatant, while no green fluorescence was observed in HeLa cells. These cells were identified as positive clones that could be subcloned.

[0096] (4) Construction of hybridoma cell lines

[0097] Subclonal sorting of positive hybridoma cells was performed using a limiting dilution method. After three rounds of subclonal and indirect immunofluorescence screening, 13 hybridoma cell lines stably secreting 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 were then detected and compared using an indirect ELISA method. The results are as follows: Figure 5As shown. Under identical experimental conditions, six antibody strains exhibited the highest titers. These strains were produced from hybridoma cell lines 13-2-G2, 13-3-B3, 13-3-E6, 13-5-B10, 13-5-G6, and 13-8-G2, respectively. The antibody clone numbers are 13-2-G2, 13-3-B3, 13-3-E6, 13-5-B10, 13-5-G6, and 13-8-G2, respectively. Figure 5 N was the negative control and P was the positive control. Based on the results of cell immunofluorescence assay and cell flow cytometry, the clone number of the hybridoma cell line with the best titer, the strongest specificity and the most wide range of applications was 13-8-G2.

[0098] The indirect ELISA procedure is as follows: Coat the microplate with 1 μg / mL human CD318 recombinant protein (100 μL / well) and incubate overnight at 4°C. The next day, discard the coating solution and wash 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, and 0.01 M potassium dihydrogen phosphate). Add 150 μL of 5% BSA to each well and block at 37°C for 2 hours. Discard the blocking solution and wash three times with PBST. Then, incubate with primary antibody, adding 100 μL of diluted antibody solution to each well. Dilute the antibody concentration of 12 positive hybridoma cell lines to 0.5 μg / mL with 1% BSA. Use immunized mouse serum with antibody titers as the positive control (P) and unimmunized mouse serum as the negative control (N). Incubate at 37°C for 1 hour and wash three times with PBST. Add 100 μL of PBST to each well. μL corresponds to a 1:5000 dilution After incubation with secondary antibody (H+L), wash, incubate at 37 ℃ for 1 hour, wash 3 times with PBST; add 100 μL TMB chromogenic solution to each well, develop at 37 ℃ for 10 min, add stop solution, and measure absorbance at 450 nm using a microplate reader.

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

[0100] Hybridoma cells with clone number 13-8-G2 were deposited and named hybridoma cell line 13-8-G2. The cell line was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C202499 on April 2, 2024.

[0101] Preparation Example 5: Preparation of Anti-CD318 Monoclonal Antibody

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

[0103] Three 10-week-old female Balb / c mice were prepared and injected intraperitoneally with 500 μL of Freund's incomplete adjuvant per mouse. One week later, they were injected intraperitoneally with hybridoma cell line 13-8-G2, 1~2×10 6 Cells / mouse. After 10-14 days, ascites fluid was collected based on the degree of abdominal distension in mice. The collected ascites fluid was centrifuged at 12000 rpm for 10 min at 4°C, and the clear yellow liquid at the center was collected. Antibodies were purified using Protein A / G affinity chromatography, and antibody concentration was determined using Nanodrop. Purity was identified by SDS-PAGE gel electrophoresis. SDS-PAGE gel electrophoresis results are shown below. Figure 6 As shown in the figure, the purity of the purified anti-CD318 monoclonal antibody is >95%, and IgG heavy chains with a molecular weight of 50 kDa and light chains with a molecular weight of 25 kDa are visible. M is the protein molecular weight standard; 1 and 2: anti-CD318 monoclonal antibody in the reduced state, 2 μg.

[0104] Preparation Example 6: Preparation of CD318 Enzyme-Linked Immunosorbent Assay Kit

[0105] The CD318 enzyme-linked immunosorbent assay kit includes the anti-CD318 monoclonal antibody obtained in Preparation Example 5, blocking buffer and enzyme-labeled secondary antibody, washing buffer, TMB chromogenic solution, and stop solution. The blocking buffer 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, and 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 fluorescently modified anti-CD318 monoclonal antibody, cell fixation solution, blocking solution, washing solution, and nuclear dye. The fluorescent group can be any one of FITC, Cy3, Cy5, or Alexa Fluor 488. Taking FITC labeling as an example, the procedure for modifying the anti-CD318 monoclonal antibody with a fluorescent group is as follows: FITC solution is added dropwise to the anti-CD318 monoclonal antibody solution obtained in Preparation Example 5 under magnetic stirring. After stirring continuously at room temperature (20℃±5℃) for 4-6 hours, the mixture is centrifuged. The supernatant is the FITC-labeled anti-CD318 monoclonal antibody. 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 includes the anti-CD318 monoclonal antibody obtained in Preparation Example 5, antibody dilution buffer, cell fixation buffer, washing buffer, and fluorescent secondary antibody. The fluorescent secondary antibody is... .

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

[0111] In this embodiment, 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 Proteintech mouse monoclonal antibody subtype identification kit. The specific procedure was as follows: 50 μL of the supernatant from hybridoma cell line 13-8-G2 was diluted 1:50 and added to the sample wells of an ELISA strip. 50 μL of HRP-labeled secondary antibody for each subtype was added to each well. The mixture was gently stirred and incubated at room temperature (20℃±5℃) for 1 hour. The liquid in the wells was discarded, and the plate was washed three times with 1×PBST. The plate was patted dry on absorbent paper, and the freshly prepared chromogenic solution was added to the wells. The plate was incubated at room temperature (20℃±5℃) in the dark for 10–20 min. Stop solution was added to each well, and the OD450 was read using a microplate reader. Figure 7 The results of the subtype identification of the anti-CD318 monoclonal antibody secreted by the hybridoma cell line 13-8-G2 provided in this embodiment show 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 Anti-CD318 Monoclonal Antibody

[0115] First, total RNA was extracted from the hybridoma cell line 13-8-G2 described in Example 4 using the Trizol method: 1×10⁻⁶ RNA was collected by centrifugation at 1000 rpm for 3 min. 6 Collect 1 positive hybridoma cell, 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 thoroughly by pipetting and transfer to a 1.5 mL EP tube, and incubate at room temperature for 5 min to ensure complete cell lysis. Add 200 μL of chloroform, shake vigorously to mix thoroughly, incubate at room temperature for 5 min, centrifuge at 12000 rpm for 15 min at 4 °C, and transfer approximately 300 μL of the supernatant to a new 1.5 mL EP tube. Add 0.5 mL of isopropanol, gently invert several times to mix, incubate at room temperature for 10 min, then centrifuge at 12000 rpm for 10 min at 4 °C. A gelatinous RNA precipitate will be visible at the bottom of the tube. Carefully aspirate the supernatant, add 1 mL of 75% ethanol, gently invert the centrifuge tube to resuspend the precipitate, and then centrifuge at 12000 rpm for 5 min at 4 °C. Discard the supernatant, allow the EP tube to air dry at room temperature for 5 min to allow the ethanol to evaporate completely, and dissolve the precipitate in RNase-free double-distilled water to obtain total RNA. Use a reverse transcription kit to reverse transcribe the extracted total RNA into cDNA.

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

[0133] SEQ ID NO:12

[0134] SISYSGYTFYNPSLKS

[0135] SEQ ID NO:13

[0136] SDGNYDYSMDY

[0137] Example 3: Detection of recombinant human CD318 protein using a CD318 enzyme-linked immunosorbent assay kit.

[0138] In this embodiment, the CD318 enzyme-linked immunosorbent assay kit described in Preparation Example 6 was used to detect the sample:

[0139] The purified human CD318 recombinant protein from Preparation Example 3 was used to coat an ELISA plate. 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 Invitrogen's recombinant anti-CDCP1 antibody (Human IgG), clone number 30D4 (catalog number: MA5-50097).

[0140] The specific operating steps are as follows: Coat the microplate with 1 μg / mL human CD318 recombinant protein, 100 μL / well, and incubate overnight at 4℃; the next day, discard the coating solution, wash three times with PBST, add 150 μL of 5% BSA to each well, and block at 37℃ for 2 hours; discard the blocking solution, wash three times with PBST, and then incubate with primary antibody. Dilute the anti-CD318 monoclonal antibody and control antibody 30D4 with 1% BSA to a concentration of 0.5 μg / mL, and add 100 μL of 1% BSA to each well for the negative control. Incubate at 37℃ for 1 hour, wash three times with PBST, and add 100 μL of the corresponding 1:5000 diluted solution to each well. (H+L) and After incubation with secondary antibody (H+L), wash and incubate at 37°C for 1 hour. Wash three times with PBST. Add 100 μL of TMB chromogenic solution to each well and incubate at 37°C for 10 min. After adding stop solution, measure the absorbance at 450 nm using a microplate reader.

[0141] The results of the ELISA experiment are as follows Figure 9 As shown, 1: anti-CD318 monoclonal antibody; 2: negative control; 3: positive control antibody 30D4. The anti-CD318 monoclonal antibody secreted by 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 the affinity constant of anti-CD318 monoclonal antibody

[0143] This embodiment describes the determination of the affinity constant of the anti-CD318 monoclonal antibody described in Preparation Example 5.

[0144] The recombinant human CD318 protein obtained in Example 3 was used to coat an ELISA plate with 100 μL of the protein in 1 μg / mL well, and incubated overnight at 4°C. The coating solution was discarded the next day, and the plate was washed three times with PBST. 150 μL of 5% BSA was added to each well, and the plate was blocked at 37°C for 2 hours. After discarding the blocking solution, the plate was washed three times with PBST and then incubated with primary antibody. The monoclonal antibody purified in Example 5 (initial concentration of 0.5 mg / mL) was serially diluted 2-fold starting from 1:200, for a total of 13 dilutions. One well was left as a blank control. 100 μL of the antibody was added to each well, and the plate was incubated at 37°C for 1 hour. The plate was then washed three times with PBST. (H+L) 1:5000 dilution, add 100 μL to each well, incubate at 37℃ for 1 hour, wash 3 times with PBST; add 100 μL TMB chromogenic solution to each well, incubate at 37℃ for 10 min, add stop solution, and measure the absorbance at 450 nm using a microplate reader. The results are shown in Table 2. Find the dilution factor A corresponding to half of the maximum binding OD value, and calculate the affinity constant using the following formula: Affinity constant = (150000 × A) / original antibody concentration.

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

[0146]

[0147] Using the above formula, the affinity constant of the anti-CD318 monoclonal antibody described in this invention is calculated to be approximately 7.68 × 10⁻⁶. 9 The present invention provides an anti-CD318 monoclonal antibody that has a high affinity for the CD318 protein.

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

[0149] This embodiment uses the CD318 immunofluorescence detection kit described in Preparation Example 7 to detect various cell types.

[0150] HeLa cells, HeLa-CD318 cells, A549 cells, NCI-H460 cells, HT-29 cells, and HCT116 cells were seeded into 24-well plates containing cell spreaders and cultured overnight. The cells were 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, and 0.01 M potassium dihydrogen phosphate), fixed with 4% paraformaldehyde for 15 min, washed three times with PBS buffer, and blocked at 37°C for 2 hours with 5% BSA solution. After discarding the blocking solution, the cells were washed three times with PBS buffer and then incubated with primary antibody. 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 for nuclear staining, wash three times with PBS buffer, blot dry the slide with absorbent paper, mount with mounting medium containing anti-fluorescence quencher, observe and photograph under a fluorescence microscope. Results are as follows: Figure 10 As shown. By Figure 10 It is understood that the anti-CD318 monoclonal antibody provided by the present invention can specifically recognize the CD318 protein.

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

[0152] This embodiment uses the flow cytometry detection kit described in Preparation Example 8 to detect various cell types.

[0153] HeLa cells, HeLa-CD318 cells, A549 cells, NCI-H460 cells, HT-29 cells, and HCT116 cells were treated to obtain single-cell suspensions. Anti-CD318 monoclonal antibody diluted in PBS buffer containing 3% BSA was added, and the cells were incubated at room temperature (20℃±5℃) or 4℃ for 30 min. The cells were washed three times with ice-cold PBS buffer, and then resuspended with fluorescent secondary antibody diluted in PBS buffer containing 3% BSA. The cells were then incubated at room temperature (20℃±5℃) or 4℃ in the dark for 30 min. The cells were washed three times with ice-cold PBS buffer, and then resuspended in PBS buffer. Cell analysis was performed using flow cytometry. If immediate flow cytometry was not possible, cells could be resuspended in 1% paraformaldehyde and stored at 4℃, but this process should not exceed 16 hours. Detection results are as follows: Figure 11 As shown.

[0154] Depend on Figure 10It was found that the antibody hardly bound to the membrane surface antigen of HeLa cells (CD318 negative), but bound well to the membrane surface antigens of other CD318 positive cells. The binding rates to the membrane surface antigens of HeLa-CD318 cells, A549 cells, NCI-H460 cells, HT-29 cells, and HCT116 cells were 94.3%, 64.2%, 46.0%, 100.0%, and 98.5%, respectively. This invention provides an anti-CD318 monoclonal antibody that specifically recognizes the CD318 protein, and it has significant application potential in the preparation of CD318 detection kits.

[0155] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

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

2. The use of the hybridoma cell line 13-8-G2 as described in claim 1 in the preparation of anti-CD318 monoclonal antibody.

3. An anti-CD318 monoclonal antibody, characterized in that, Includes variable regions for light chains and variable regions for heavy chains; The light chain variable region includes CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO: 6-8; The heavy chain variable region includes CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO: 11-13.

4. The anti-CD318 monoclonal antibody as described in claim 3, characterized in that, The anti-CD318 monoclonal antibody is secreted by the hybridoma cell line 13-8-G2 or its passaged cell line as described in claim 1.

5. A nucleic acid molecule encoding the anti-CD318 monoclonal antibody as described in claim 3 or 4.

6. The method for preparing the anti-CD318 monoclonal antibody as described in claim 3 or 4, characterized in that, include: Mice were immunized with an incomplete adjuvant for 1-2 weeks, and then injected intraperitoneally with 1-2 × 10⁻⁶ mg / L. 6 The hybridoma cell line 13-8-G2 with accession number CCTCC NO: C202499 was used. Mouse ascites fluid was collected after 10-14 days, purified, and the anti-CD318 monoclonal antibody was obtained.

7. The use of the anti-CD318 monoclonal antibody as described in claim 3 or 4 in any of the following: (I) Preparation of immunoassay products for detecting CD318 protein; and / or (II) Preparation of products targeting the CD318 protein; and / or (III) Preparation of products for labeling tumor cells.

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

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

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

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