Mouse anti-human ctrb1 monoclonal pair antibody and hybridoma cell strain secreting the same

Abstract

The application provides a mouse anti-human CTRB1 monoclonal pairing antibody and a hybridoma cell strain secreting the antibody, and relates to the fields of bioengineering and biomedical technology. The hybridoma cell strains secreting the CTRB1 monoclonal antibody have clone numbers of 3D5-1 and 4G5-3 respectively, and preservation numbers of CCTCC NO: C202491 and CCTCC NO: C202490 respectively. The application contains a monoclonal antibody generated by the hybridoma cell strain 3D5-1, and the antibody comprises a heavy chain variable region and a light chain variable region. The application also contains a monoclonal antibody generated by the hybridoma cell strain 4G5-3, and the antibody comprises a heavy chain variable region and a light chain variable region. The monoclonal antibodies generated by 3D5-1 and 4G5-3 can be used for detecting CTRB1 expression conditions in scientific research or clinical immunohistochemistry, immunoblotting and the like, and can also be used in pairs to detect CTRB1 content through enzyme-linked immunoassay, chemiluminescence and the like.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and biomedical technology, specifically to a mouse anti-human CTRB1 monoclonal paired antibody and a hybridoma cell line that secretes the antibody. Background Technology

[0002] According to the Global Cancer Statistics Yearbook, in 2020, there were nearly 1.1 million new cases of stomach cancer worldwide (5.6%), and 770,000 deaths (7.7%). Stomach cancer ranked fifth in new cases and fourth in deaths among the 36 types of cancer surveyed. It is noteworthy that stomach cancer presents a very serious threat, being a highly prevalent malignant disease impacting national health and economic development.

[0003] However, currently, basic and clinical research on the diagnosis and treatment of gastric cancer in my country remains relatively underdeveloped, mainly in two aspects: First, the course of gastric cancer (including multiple stages such as chronic gastritis, intestinal metaplasia, atypical hyperplasia, carcinoma in situ, and metastatic cancer) is insidious and often difficult to detect, relying primarily on electronic endoscopy and pathological examination. However, the adoption rate of electronic endoscopy in my country is still low, and patient compliance is also poor, resulting in a low early diagnosis rate for gastric cancer. Second, the treatment of gastric cancer mainly includes surgical treatment, radiotherapy, chemotherapy, and targeted therapy. Chemotherapy is the primary treatment method for patients with advanced or metastatic gastric cancer. However, there is a lack of highly sensitive treatment and prognostic biomarkers for different treatment regimens of gastric cancer, which leads to poor clinical prognosis and quality of life for patients.

[0004] Therefore, finding effective biomarkers for the early diagnosis, treatment, and prognosis of gastric cancer is of great significance for the early differential diagnosis and timely and appropriate treatment of gastric cancer. In terms of diagnosis, there is currently a lack of effective non-invasive early screening and auxiliary diagnostic biomarkers for gastric cancer. Although gastroscopy is the gold standard for gastric cancer diagnosis, the rate of gastroscopy in my country is relatively low due to poor patient compliance. As a result, approximately 90% of newly diagnosed gastric cancer patients are in the advanced stage, thus missing the opportunity for minimally invasive treatment or radical surgery. This is also one of the important reasons for the high mortality rate of gastric cancer in my country.

[0005] Furthermore, due to the lack of specific non-invasive screening biomarkers for gastric cancer, clinical practice still relies on traditional serum diagnostic indicators such as CEA, CA153, CA199, CA242, CA50, CA724, and PG for gastric cancer screening, which leads to high misdiagnosis rates and false negatives. In terms of treatment, for patients with advanced gastric cancer, chemotherapy remains the primary treatment, supplemented by targeted therapies. This is mainly because there are currently few available targeted drugs and the target population is limited. For example, most gastric cancer patients are Her2 negative and unlikely to benefit from Herceptin. In addition, although immunotherapy, represented by PD-1 antibodies, has been approved for clinical treatment of gastric cancer, it is primarily targeted at patients with microsatellite instability and EBV-positive gastric cancer, which account for approximately 10-15% of all gastric cancer cases.

[0006] More importantly, compared to other types of tumors, the development of novel anti-tumor strategies has progressed very slowly. On the one hand, the small number of gastric cancer patients in developed Western countries reduces the incentive for research and development targeting gastric cancer. On the other hand, the highly heterogeneous nature of gastric cancer means that targeted strategies effective in other cancers have little effect in gastric cancer treatment. For example, the anti-angiogenic drug Avastin and the anti-EGFR drug Erbitux are very effective in colorectal cancer, but largely ineffective in gastric cancer. Furthermore, due to the scarcity of patients with DNA-damaged tumors, clinical studies of PARP inhibitors in gastric cancer have largely failed.

[0007] In summary, these realities compel us to urgently analyze the patterns of gastric cancer development from new research perspectives and explore novel treatment strategies for gastric cancer using innovative approaches. Therefore, identifying effective biomarkers for early diagnosis, auxiliary diagnosis, treatment, and prognosis of gastric cancer is of great significance for early differential diagnosis and timely and appropriate treatment of gastric cancer.

[0008] Chymotrypsinogen B (CTRB1) is a precursor of pancreatic proteases. Synthesized in pancreatic acinar cells and secreted into the small intestine, it is hydrolyzed and activated to produce functional enzymes. CTRB1 activates serine endopeptidase activity, participating in responses to apoptotic signals, nutrients, cytokines, and peptide hormones. Using a combination of 4D Label-free quantitative techniques and PEA technology on the Olink platform, CTRB1 protein was found to be associated with the development and progression of gastric cancer. Subsequent molecular and cellular biological mechanism studies confirmed that CTRB1 protein has the potential to develop a simple, rapid, non-invasive, low-cost, and easily dynamic monitoring technology for the development and progression of gastric cancer, which is of great significance for the comprehensive diagnosis and treatment of gastric cancer.

[0009] Therefore, there are areas for improvement. The present invention provides a mouse anti-human CTRB1 monoclonal paired antibody and a hybridoma cell line that secretes the antibody. Summary of the Invention

[0010] To address the shortcomings of existing technologies, the present invention aims to propose a mouse anti-human CTRB1 monoclonal paired antibody and a hybridoma cell line that secretes this antibody. The specific scheme is as follows:

[0011] A hybridoma cell line that secretes a mouse anti-human CTRB1 monoclonal paired antibody comprises:

[0012] Hybridoma cell line 3D5-1, accession number CCTCC NO:C202491;

[0013] Hybridoma cell line 4G5-3, accession number CCTCC NO:C202490;

[0014] Hybridoma cell lines 3D5-1 and 4G5-3 used the CTRB1 protein expressed by mammalian cells as an antigen to secrete CTRB1 monoclonal antibodies.

[0015] A mouse anti-human CTRB1 monoclonal paired antibody, the paired antibody comprising a monoclonal antibody produced by the hybridoma cell line 3D5-1 and a monoclonal antibody produced by the hybridoma cell line 4G5-3.

[0016] Furthermore, the monoclonal antibodies produced by the hybridoma cell line 3D5-1 include both heavy chain variable regions and light chain variable regions;

[0017] The amino acid sequence of the heavy chain variable region is SEQ ID No:1;

[0018] The amino acid sequence of the light chain variable region is SEQ ID No:2.

[0019] Furthermore, the monoclonal antibodies produced by the hybridoma cell line 4G5-3 include both heavy chain variable regions and light chain variable regions;

[0020] The amino acid sequence of the heavy chain variable region is SEQ ID No:3;

[0021] The amino acid sequence of the light chain variable region is SEQ ID No:4.

[0022] Furthermore, the amino acid sequence SEQ ID No:1 of the heavy chain variable region is encoded by a DNA molecule with the sequence SEQ ID No:5.

[0023] Furthermore, the amino acid sequence of the light chain variable region is SEQ ID No:2, encoded by a DNA molecule with sequence SEQ ID No:6.

[0024] Furthermore, the amino acid sequence SEQ ID No:3 of the heavy chain variable region is encoded by a DNA molecule with the sequence SEQ ID No:7.

[0025] Furthermore, the amino acid sequence of the light chain variable region is SEQ ID No:4, encoded by a DNA molecule with sequence SEQ ID No:8.

[0026] A method for detecting CTRB1 protein content using the aforementioned mouse anti-human CTRB1 monoclonal paired antibody, characterized in that the method comprises: detecting the CTRB1 protein content in blood using enzyme-linked immunosorbent assay (ELISA) or chemiluminescence immunoassay based on the paired antibody.

[0027] Application of the described detection method in gastric cancer screening, auxiliary diagnosis, and companion diagnosis.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] Using the hybridoma cell lines 3D5-1 and 4G5-3 of this invention, CTRB1 monoclonal antibodies can be prepared. The monoclonal antibodies produced by the hybridoma cell lines 3D5-1 and 4G5-3 can be used alone for scientific research or clinical immunohistochemistry, immunoblotting, etc. to detect CTRB1 expression. They can also be used in pairs to detect CTRB1 protein content by enzyme-linked immunosorbent assay (ELISA), chemiluminescence, etc., to assist in the comprehensive diagnosis and treatment of gastric cancer. Attached Figure Description

[0030] Figure 1 This is a graph showing the SDS-PAGE analysis results of the paired antibodies in this invention;

[0031] Figure 2 This is an electrophoretic detection result of PCR amplification of the variable regions of two monoclonal antibodies according to the present invention;

[0032] Figure 3 This is a standard curve diagram of the CTRB1 protein concentration detected by enzyme-linked immunosorbent assay (ELISA) in this invention.

[0033] Figure 4 This is a standard curve of CTRB1 protein concentration detected by chemiluminescence method in this invention;

[0034] Figure 5 This invention demonstrates the distribution of CTRB1 protein and its ability to distinguish indicators in different populations.

[0035] The preservation information is as follows:

[0036] Hybridoma cell line 3D5-1, accession number CCTCC NO:C202491, is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on March 26, 2024.

[0037] Hybridoma cell line 4G5-3, accession number CCTCC NO:C202490, is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on March 26, 2024. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0039] The purpose of this invention is to provide a pair of highly affinity, pairable mouse anti-human CTRB1 monoclonal antibodies and a hybridoma cell line that secretes these paired antibodies, to develop a method for detecting CTRB1 levels in human blood using the secreted paired antibodies, and to explore its applications in gastric cancer screening, auxiliary diagnosis, and companion diagnostics. To achieve the above objectives, this invention provides the following technical solution:

[0040] (1) The full-length CTRB1 sequence (NM_001906.6) was synthesized, and the recombinant expression vector pCDNA3.4-CTRB1 was constructed. Endotoxin-free recombinant plasmids were prepared and transfected into HEK293 cells to obtain the target protein. Classic techniques such as mouse immunization, cell fusion, subclonal selection, and pairing selection were used to prepare CTRB1 paired monoclonal antibodies. Antibody proteins 3D5-1 and 4G5-3 were purified by affinity chromatography, and antibody concentrations were determined by SDS-PAGE. The titer of the purified antibodies was determined by ELISA.

[0041] (2) Based on the constant region sequences of 3D5-1 and 4G5-3 antibodies, specific primers were synthesized, and the variable regions of the heavy chain and light chain of the antibodies were amplified by PCR. After the target fragment was recovered, cloned into the vector, transformed into E. coli, screened for positive clones, and plasmids were extracted, the heavy chain and light chain variable region sequences of monoclonal antibodies 3D5-1 and 4G5-3 were obtained.

[0042] (3) Develop detection methods and kits for human blood CTRB1 using protein-linked immunosorbent assay (sandwich method) and chemiluminescence assay (direct luminescence method) with 3D5-1 and 4G5-3 paired antibodies, and verify the detection performance of the kits.

[0043] (4) Through prospective and retrospective cohort studies, we will explore the role of CTRB1 in early screening, auxiliary diagnosis, and companion diagnosis of gastric cancer. At the same time, we will combine CTRB1 with traditional gastric cancer tumor markers (CEA, CA199, CA242, PG1 / PG2, G17, etc.) to develop an integrated scheme for gastric cancer multi-marker combination, screening, and companion diagnosis based on the characteristics of the Chinese population.

[0044] The hybridoma cell line provided by this invention, which secretes CTRB1 monoclonal antibodies by immunizing mice with CTRB1 protein expressed in mammalian cells as an antigen, is named 3D5-1 and classified as a mouse anti-human CTRB1 hybridoma cell line. This cell line has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C202491. The monoclonal antibody produced by this hybridoma cell line is of the IgG2a subtype. Its heavy chain variable region amino acid sequence is SEQ ID No:1, and its heavy chain variable region DNA sequence is SEQ ID No:5, where SEQ ID No:1 is encoded by the DNA molecule of SEQ ID No:5. Its light chain variable region amino acid sequence is SEQ ID No:2, and its light chain variable region DNA sequence is SEQ ID No:6, where SEQ ID No:2 is encoded by the DNA molecule of SEQ ID No:6.

[0045] In addition, a hybridoma cell line secreting a monoclonal paired antibody against CTRB1 was obtained, named 4G5-3, and classified as a mouse anti-human CTRB1 hybridoma cell line. This cell line has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:C202490. The monoclonal antibody produced by this cell line is of the IgG2b subtype. Its heavy chain variable region amino acid sequence is SEQ ID No:3, and its heavy chain variable region DNA sequence is SEQ ID No:7, with SEQ ID No:3 encoded by the DNA molecule of SEQ ID No:7; the light chain variable region amino acid sequence is SEQ ID No:4, and its light chain variable region DNA sequence is SEQ ID No:8, with SEQ ID No:4 encoded by the DNA molecule of SEQ ID No:8.

[0046] In some embodiments of the present invention, the anti-CTRB1 antibody further includes an Fc domain selected from the same type of IgG1, IgG3, IgG4, IgA1, IgA2, IgM, and IgE, including monoclonal antibodies, chimeric antibodies, humanized antibodies, or bispecific antibodies.

[0047] The monoclonal antibodies produced by the hybridoma cell line 3D5-1 and the monoclonal antibodies produced by the hybridoma cell line 4G5-3 constitute the anti-human CTRB1 monoclonal paired antibody.

[0048] Based on the above, the present invention proposes the following embodiments.

[0049] Example 1: Preparation and purification of anti-CTRB1 antibody

[0050] (1) Hybridoma cell preparation: Recombinant human CTRB1 protein (obtained from HEK293 cell line) was used as the immunogen. The immunogen was dissolved in PBS and mixed evenly with an equal volume of Freund's complete adjuvant to form a water-in-oil emulsion. The primary immunization was performed subcutaneously at multiple sites, with an immunization dose of 100 μg / mouse. Three weeks after the primary immunization, an equal volume of incomplete Freund's adjuvant was mixed with the immunogen and emulsified, and the mice were immunized using the same route and dose. A booster immunization was performed three weeks later to bring the antibody titer in the mouse serum to over 10,000. One week after the final booster immunization, blood was collected from the eyeballs, and the serum titer was determined by ELISA.

[0051] If the mouse serum titer does not reach 10,000, several booster immunizations must be administered. Mice with a titer of 10,000 or higher should receive a pulse immunization three days before fusion, using unadjuvanted antigen injected via the tail vein. 10–14 days before fusion, resuscitate mouse myeloma cells SP2 / 0 and culture them in DMEM medium containing 10% FCS. 24–36 hours before fusion, adjust the cell concentration to 3 × 10⁻⁶ cells using fresh medium. 5 / mL. Preheat DMEM and PEG solutions to 37°C in a water bath before fusion. Collect 2×10⁻⁶ mL. 7 One well-growing SP2 / 0 cell was washed twice with DMEM and then mixed with (1-2) × 10⁻⁶ cells. 8 Each spleen cell was thoroughly mixed in a 50 mL centrifuge tube, washed once with DMEM, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were gently loosened.

[0052] Place the centrifuge tubes in a 37°C water bath and preheat for 5 minutes. Then, pipette 1 mL of pre-warmed 50% PGE solution and gently insert the pipette into the bottom of the cells. Add the fusion agent at a uniform rate over 1 minute, stirring constantly. Let the mixture stand for 1 minute. Next, add 40 mL of pre-warmed DMME at 37°C, adding slowly at first and then quickly, stirring gently. Let the mixture stand at 37°C for 5–10 minutes. After centrifuging at 800 rpm for 10 minutes, discard the supernatant and resuspend the cells in 55–60 mL of pre-warmed 37°C DMEM medium containing 1% HAT and 20% FCS.

[0053] After gently mixing, add 60 μL to each well of a 96-well plate containing cultured cells. Incubate at 37°C with 5% CO2 for 4–5 days, then replace half the medium. When cell clones have filled 1 / 3 of the culture wells, aspirate the culture supernatant and use indirect ELISA to screen for positive hybridoma cell lines. Use limiting dilution to screen for monoclonal positive hybridoma cell lines, and simultaneously expand the positive hybridoma cells through culture and cryopreserve them for seed production.

[0054] (2) Indirect ELISA titer detection: Dilute CTRB1 antigen to 0.25 μg / mL with PBS coating buffer, mix well, and add 100 μL to each well of the ELISA strip. Incubate overnight at 4°C. After coating, discard the coating buffer, wash the plate 3 times, add 200 μL of blocking buffer to each well, and incubate at 37°C for 1 hour. Remove the ELISA plate, discard the internal solution, and wash the plate once. For monoclonal antibody, start at 1.0 μg / mL and serially dilute 2-fold, 100 μL per well, and incubate at 37°C for 1 hour. Remove the ELISA plate, discard the internal solution, wash the plate 3 times, and add 100 μL of diluted enzyme-labeled secondary antibody (goat anti-mouse-HRP, 1:20000) to each well. Incubate at 37°C for 1 hour. Remove the microplate, discard the internal solution, and wash the plate four times. Add 100 μL of TMB chromogenic buffer to each well, adjusting the development time according to the color intensity (generally 37℃, 15 min). Add 100 μL of 1M HCl solution to each well to stop the reaction. Immediately read the OD value at 450 nm on the microplate reader. The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is defined as the titer of that sample.

[0055] (3) Antibody pairing detection: Dilute the CTRB1 monoclonal antibody with the required titer to a concentration of 1 μg / mL with PBS coating buffer, add 100 μL to each well, and incubate overnight at 4°C. After coating, discard the coating buffer, wash the plate 3 times, add 200 μL of blocking buffer to each well, and incubate at 37°C for 1 h. After blocking, wash 3 times with 1×PBST (PBS + 0.05% Tween-20), 200 μL / well each time, and let stand for 30 s each time. Start with CTRB1 protein 50 ng / mL, dilute 2-fold, add 100 μL to each well, and incubate at 37°C for 1 h. Remove the microplate, discard the internal solution, wash the plate 3 times, add 100 μL of mouse monoclonal antibody-HRP with the required titer (1 / 5000 dilution) to each well, and incubate at 37°C for 1 h. Remove the microplate, discard the internal solution, wash the plate four times, and add 100 μL of TMB chromogenic solution to each well. The chromogenic time depends on the color intensity, generally 10-15 min at 37℃. Add 100 μL of 1M HCl solution to each well to stop the reaction. Immediately take a reading at 450 nm on the microplate reader.

[0056] (4) Antibody preparation and purification: 7–10 days before hybridoma cell inoculation, Balb / c mice should be intraperitoneally injected with 0.5 mL of toluene (or liquid paraffin). Hybridoma cell inoculation volume: 10 5 ~10 7 Cells per mouse were injected into the peritoneal cavity of the mice, and ascites fluid was collected within 1–3 weeks. The collected supernatant was centrifuged at high speed, filtered under vacuum through a microporous membrane, and purified using a protein A / G column to obtain the corresponding antibodies.

[0057] Results: Through the above steps, hybridoma cell lines 3D5-1 and 4G5-3 capable of secreting CTRB1 monoclonal antibodies were obtained. The secreted antibodies met the detection requirements for both titer and antibody pairing assays. The analysis results of the paired antibodies after antibody preparation and purification are shown in the table below. Figure 1 .

[0058] from Figure 1 -A and Figure 1 -B results show that Lane M, Lane R, and Lane NR all exhibit consistent performance. Implementation Case 2: Variable region sequence analysis of monoclonal antibodies 3D5-1 and 4G5-3.

[0059] Total RNA was extracted from the cultured hybridoma cell lines 3D5-1 and 4G5-3 respectively: (1) Tissue homogenization: Add sufficient TRIPure Reagent to the cell sample and repeatedly pipette. (2) Place the above homogenate at room temperature for 5 min to allow nucleic acids and proteins to fully dissociate. Add 0.2 mL of chloroform to each 1 mL of TRIPure Reagent, tighten the cap, shake vigorously manually for 15 s, and then let stand at room temperature for 2-3 min. (3) Centrifuge at 12000 g for 10 min at 4℃. (4) Carefully aspirate the upper aqueous phase (colorless) into a new test tube and calculate the volume of the aspirated aqueous phase. (5) Add an equal volume of pre-cooled isopropanol to the aspirated aqueous phase, tighten the cap, and shake gently. (6) Let stand at room temperature for 10 min to allow RNA to fully precipitate. (7) Centrifuge at 12000 g for 10 min at 4℃. (8) Discard the supernatant (be careful not to discard the precipitate), add 1 mL of 80% ethanol to each tube to rinse, tighten the cap, and gently shake the centrifuge tube to remove residual isopropanol and salt. Centrifuge at 7500g for 5 min at 4℃. (9) Discard the supernatant, open the cap, and dry the RNA precipitate (evaporate at room temperature or vacuum dry). Note that the RNA precipitate should not be completely dried, otherwise it will be difficult to dissolve. (10) Dissolve the RNA precipitate in an appropriate amount of RNase-free ddH2O. Take 1 μL, perform 1% agarose gel electrophoresis, and stain with GelStain. Determine the RNA concentration and purity. Reverse transcribe the extracted RNA into cDNA and perform PCR amplification using specific primers (see...). Figure 2 The product was then TA cloned and sequenced.

[0060] Through the above steps (1)-(10), the hybridoma cell line and antibody subtype were successfully identified. The sequences involved in this invention are as follows:

[0061] SEQ ID No:1 (3D5-1 heavy chain variable region amino acid sequence)

[0062] EVQLEQSGAELVRPGASVRLSCKASGFSFTNYWLHWVKQRPGQGLEWIGMI

[0063] HPSDSETRLNQKFMDKATLTVDKSSSTAYMQLRSPTSEDSAVYYCARDYSGYA

[0064] YFDVWGAGTTVTVSS

[0065] SEQ ID No:2 (3D5-1 light chain variable region amino acid sequence)

[0066] DIVMTQSPVSLAVSLGQRATISCRASESVDRYGKSIMHWYQQKPGQPPKLLIY

[0067] GIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSSEDPPTFGGGTKLEIK

[0068] SEQ ID No:3 (4G5-3 heavy chain variable region amino acid sequence)

[0069] EVQLQESGPSLVKPSQTLSLTCSVTGDSITSDYWNWIRRFPGNKLEYLGFLRYS

[0070] GGTYYNPSLKSRISITRDPSKNQYYLQLNSVTTEDTATYYCARHGNSWYFDV

[0071] WGAGTTVTVSSSEQ ID No:4 (4G5-3 light chain variable region amino acid sequence)

[0072] DIVMTQSTASLAVSLGQRATISCRASESVDSFGNSFMHWYQQKPGQPPKLLIY

[0073] RASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEAPPTFGGGTK

[0074] LEIKSEQ ID No:5 (3D5-1 heavy chain variable region DNA sequence)

[0075] GAGGTGCAGCTGGAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGAGCTTC

[0076] AGTGAGGCTGTCCTGCAAGGCTTCTGGCTTCTCCTTCACCAACTACTGGCT

[0077] GCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGCATGA

[0078] TTCATCCTTCCGATAGTGAAACTAGGTTAAATCAGAAGTTCATGGACAAGG

[0079] CCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGCAACTCCGCA

[0080] GCCCGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGATTACTCCG

[0081] GCTACGCGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCT

[0082] CAGSEQ ID No:6 (DNA sequence of the variable region of the 3D5-1 light chain)

[0083] GATATTGTGATGACCCAGTCTCCAGTTTCTTTGGCTGTGTCTCTAGGGCAGA

[0084] GGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATCGTTATGGCAAGA

[0085] GTATTATGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCA

[0086] TCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAGTGGCA

[0087] GTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGCTGATG

[0088] ATGTTGCAACCTATTACTGTCAGCAAAGTAGTGAGGATCCTCCGACGTTCG

[0089] GTGGAGGCACCAAGCTGGAAATCAAACSEQ ID No:7 (DNA sequence of the variable region of the 4G5-3 heavy chain)

[0090] GAGGTCCAGCTGCAGGAGTCAGGACCTAGCCTCGTGAAACCTTCTCAGAC

[0091] TCTGTCCCTCACCTGTTCTGTCACTGGCGACTCCATCACCAGTGATTACTGG

[0092] AACTGGATCCGGAGATTCCCAGGGAATAAACTTGAATACCTGGGGTTCTTG

[0093] AGGTACAGTGGTGGCACTTACTACAATCCATCTCTCAAAAGTCGAATCTCC

[0094] ATCACTCGAGACCCATCCAAGAACCAGTACTACCTGCAGTTGAATTCTGTG

[0095] ACTACTGAGGACACAGCCACATATTACTGTGCAAGACATGGTAACTCCTGG

[0096] TACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGSEQ ID No:8 (DNA sequence of the variable region of the 4G5-3 light chain)

[0097] GATATTGTGATGACACAATCTACAGCTTCTTTGGCTGTGTCTCTAGGACAGA

[0098] GGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTTTGGCAATA

[0099] GTTTTATGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCA

[0100] TCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAGTGGCA

[0101] GTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGCTGATG

[0102] ATGTTGCAACCTATTACTGTCAGCAAAGTAATGAGGCTCCTCCGACGTTCG

[0103] GTGGAGGCACCAAGCTGGAAATCAAAC

[0104] Example 3: Development of a human blood CTRB1 protein-linked immunosorbent assay (sandwich method) and chemiluminescence (direct luminescence) detection method

[0105] This section describes the development of a detection method and kit for detecting CTRB1 protein levels in human blood using purified paired mouse monoclonal antibodies 3D5-1 and 4G5-3 (i.e., paired antibodies).

[0106] The detection methods used are enzyme-linked immunosorbent assay (ELISA) or chemiluminescence immunoassay (chemiluminescence immunoassay), as detailed below:

[0107] (1) Enzyme-linked immunosorbent assay (ELISA): Dilute 3D5-1 mouse monoclonal antibody to the required concentration with PBS coating buffer, mix well, and add 100 μL to each well of the strip. Incubate overnight at 4°C. After coating, discard the coating buffer, wash the plate three times, add 200 μL of blocking buffer to each well, and incubate at 37°C for 1 h. Remove the ELISA plate, discard the inner solution, and wash the plate once. Start with 50 ng / mL of CTRB1 protein, dilute 2-fold, add 100 μL to each well, and incubate at 37°C for 1 h. Remove the ELISA plate, discard the inner solution, wash the plate three times, and add 100 μL of diluted 4G5-3 mouse monoclonal antibody-HRP to each well (4G5-3 mouse monoclonal antibody-HRP: 1 / 5000 dilution). Incubate at 37°C for 1 h. Remove the microplate, discard the internal solution, wash the plate four times, and add 100 μL of TMB chromogenic solution to each well. The chromogenic time depends on the color intensity, generally 10-15 min at 37℃. Add 100 μL of 1M HCl solution to each well to stop the reaction. Immediately take a reading at 450 nm on the microplate reader.

[0108] Results: The enzyme-linked immunosorbent assay (ELISA) method developed using paired antibodies 3D5-1 and 4G5-3 showed a good linear relationship between CTRB1 protein concentration and absorbance at A450 nm in the range of 0–50 ng / mL. (See attached image.) Figure 3 .

[0109] (2) Chemiluminescence method: The 3D5-1 antibody was coated with toluenesulfonyl magnetic beads: Taking 15mg of magnetic beads per tube as an example, 0.15mL of the original magnetic bead solution was magnetically separated in a coating tube. 0.75mL of magnetic bead coating solution was added to resuspend the magnetic beads 1#, and after mixing, magnetic separation was performed and the supernatant was discarded. 0.25mL of magnetic bead coating solution 4# was added, followed by 0.125mL of magnetic bead coating solution 1#, and the mixture was stirred evenly to obtain a magnetic bead suspension. 0.015mL of the protein solution to be coated was placed in a clean centrifuge tube, and 0.36mL of magnetic bead activation solution 1# was added and stirred evenly to obtain a diluted protein solution. The diluted protein solution was added to the above magnetic bead suspension, mixed evenly, and then placed on a blood mixer in an incubator. The mixture was rotated and mixed at 37℃ for 24h, and the magnetic bead coating concentration was 20mg / mL. Remove the coupled magnetic beads from the incubator, perform magnetic separation, discard the supernatant, add 0.125 mL of magnetic bead blocking buffer #4 to resuspend the magnetic beads, perform magnetic separation again, and discard the supernatant. Then resuspend again with 0.125 mL of magnetic bead blocking buffer #4 and place on a blood mixer in the incubator, rotating and mixing at 37°C for 24 hours. Remove the blocked magnetic beads from the incubator, perform magnetic separation, and discard the supernatant. Add 0.125 mL of magnetic bead blocking buffer #2 to resuspend the magnetic beads, perform magnetic separation again, discard the supernatant, and add another 0.125 mL of magnetic bead blocking buffer #2 to resuspend again. The magnetic bead storage concentration is 20 mg / mL. After coating, tighten the tube cap, affix a label, and store at 2–8°C.

[0110] Acridinium ester labeling of 4G5-3 antibody: Taking the preparation of 0.2 mg antibody-acridinium ester label as an example, the antibody:acridinium ester labeling molar ratio is 1:10, the antibody molecular weight is calculated as 150 kDa, and 1.667 μL of OC023 is added. CTRB1: 148 kDa, and the volume of OC023 used is 1.69 μL. Take one desalting column, remove the bottom seal, put it into a 2 mL centrifuge tube, and centrifuge to remove the liquid in the tube. Add 300 μL of labeling buffer 6#, centrifuge at 1500g for 1 min, remove the liquid in the centrifuge tube, and repeat 6 times. Take 130 μL of protein and add it to the desalting column for desalting once, collect the protein (centrifuge parameters: 1500g, 2 min). Use a NanoDrop 2000 micro-volume spectrophotometer (Protein A280, Type selected as IgG) with labeling buffer 6# as Blank to detect the concentration. Dilute the protein to be labeled to 2 mg / mL with labeling buffer #6 according to the measured concentration. Add 100 μL of protein to a 1.5 mL centrifuge tube, add 1.69 μL of OC023 to the protein and mix quickly. Incubate at 25°C in the dark for 60 min. Add 1.315 μL of labeling blocking agent #1 to a 1.5 mL protein labeling reaction tube, mix quickly, and incubate at 25°C in the dark for 30 min. Take two desalting columns and pre-wash them 6 times with labeling buffer #7, then discard the buffer (centrifuge parameters: 1500g, 1 min). After the reaction is complete, add the labeled protein to the desalting column and desalt twice consecutively (centrifuge parameters: 1500g, 2 min). Collect the protein in a 1.5 mL centrifuge tube. For the second collection, the centrifuge tubes were weighed. After centrifugation, the mass of the centrifuge tube containing the protein label was measured, and the concentration was determined using a NanoDrop 2000 micro-spectrophotometer (Protein A280, Type selected IgG) with labeling buffer 7# as Blank. Based on the measured concentration, the protein to be labeled was diluted to 1 mg / mL with labeling buffer 7#. Additives 1# and 2# were then added and mixed thoroughly to obtain an acridine ester label with a concentration of 0.5 mg / mL, which was stored at -20℃.

[0111] Chemiluminescence detection system: 250 μL of 3D5-1-toluenesulfonyl magnetic beads (20 mg / mL) was diluted with antibody diluent to 0.5 mg / mL in 10 mL volume, which can be used to detect 200 samples. 5 μL of 4G5-3-acridinyl ester label (0.5 mg / mL) was diluted 1:2000 with antibody diluent in 10 mL volume, which can be used to detect 200 samples. Based on the detection results of Nanoorange, 5 μL of CTRB1 protein (1 mg / mL) was diluted to 1 mL volume, with a protein concentration of 4262 ng / mL. This was further diluted with antibody diluent to standard concentrations of 0, 3.330, 6.659, 13.319, 53.275, and 106.550 ng / mL. The detection parameters were set to a one-step reaction mode. 10 μL of sample was taken, 50 μL of 4G5-3-acridinyl ester label was diluted 1:2000 (Rd), and 50 μL of 3D5-1-toluenesulfonyl magnetic beads at a concentration of 0.5 mg / mL (Ra) were taken. The mixture was incubated for 15 min. The unit was ng / mL. The detection was then performed.

[0112] Results: The chemiluminescence (direct luminescence) detection method developed using paired antibodies 3D5-1 and 4G5-3 showed a good linear relationship between CTRB1 protein concentration and luminescence value in the range of 0–100 ng / mL. (See attached image.) Figure 4 .

[0113] Example 4: The role of CTRB1 protein in early screening, auxiliary diagnosis, and companion diagnosis of gastric cancer

[0114] To verify the role of CTRB1 protein in early screening, auxiliary diagnosis, and companion diagnosis of gastric cancer, we collected plasma samples from 400 healthy individuals who underwent gastroscopy and 400 gastric cancer patients (100 each from stages I, II, III, and IV). Among the gastric cancer patients, 100 samples were paired before and after standardized treatment. We also collected plasma samples from 200 patients with non-gastrointestinal tumors such as liver cancer, lung cancer, and cervical cancer. Diagnostic tests were performed using the CTRB1 protein detection method developed in Example 3. The results are shown in [Figure 3]. Figure 5 .

[0115] It can be seen that the concentration of CTRB1 protein in the blood of gastric cancer patients is significantly higher than that in healthy individuals. Figure 5 -A), and gradually increased with tumor stage progression. ROC curve analysis showed that its AUC was 0.93, P < 0.0001. Figure 5-C), suggesting that CTRB1 protein may serve as a screening indicator for gastric cancer. After selecting an appropriate threshold, its detection sensitivity was 74.57%, specificity 91.95%, positive predictive value 75.18%, and negative predictive value 70.14%, significantly higher than the detection performance of currently used clinical tumor markers. With the combined use of multiple indicators, the sensitivity can be further improved to 85.16%. Furthermore, the comparison of CTRB1 protein concentration in the blood of gastric cancer patients before and after standardized treatment shows (…). Figure 5 -B) shows a significant decreasing trend, suggesting that CTRB1 protein may serve as a companion diagnostic indicator for assessing the efficacy of gastric cancer treatment.

[0116] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A hybridoma cell line secreting anti-human CTRB 1 monoclonal pair of antibodies, characterized in that, Comprising: The hybridoma cell strain 3D5-1, the preservation number is CCTCC NO: C202491; The hybridoma cell strain 4G5-3, the preservation number is CCTCC NO: C202490; The hybridoma cell strain 3D5-1 and the hybridoma cell strain 4G5-3 respectively take the CTRB1 protein expressed by mammalian cells as an antigen, realize secreting CTRB1 monoclonal antibody.

2. Anti-human CTRB 1 monoclonal pairing antibodies, characterized in that, The pairing antibody includes the monoclonal antibody produced by the hybridoma cell strain 3D5-1 of claim 1 or the monoclonal antibody produced by the hybridoma cell strain 4G5-3 of claim 1.

3. The anti-human CTRB 1 monoclonal pairing antibody according to claim 2, characterized in that, The monoclonal antibody produced by the hybridoma cell strain 3D5-1 includes a heavy chain variable region and a light chain variable region; The heavy chain variable region amino acid sequence is SEQ ID No: 1; The light chain variable region amino acid sequence is SEQ ID No:

2.

4. The anti-human CTRB 1 monoclonal pairing antibody according to claim 2, characterized in that, The monoclonal antibody produced by the hybridoma cell strain 4G5-3 includes a heavy chain variable region and a light chain variable region; The heavy chain variable region amino acid sequence is SEQ ID No: 3; The light chain variable region amino acid sequence is SEQ ID No:

4.

5. The anti-human CTRB 1 monoclonal pairing antibody according to claim 3, characterized in that, The heavy chain variable region amino acid sequence SEQ ID No: 1 is encoded by the DNA molecule with the sequence of SEQ ID No:

5.

6. The anti-human CTRB 1 monoclonal pairing antibody of claim 3, wherein, The light chain variable region amino acid sequence SEQ ID No: 2 is encoded by the DNA molecule with the sequence of SEQ ID No:

6.

7. The anti-human CTRB 1 monoclonal pairing antibody according to claim 4, characterized by The heavy chain variable region amino acid sequence SEQ ID No: 3 is encoded by the DNA molecule with the sequence of SEQ ID No:

7.

8. The anti-human CTRB 1 monoclonal pairing antibody according to claim 4, characterized by The light chain variable region amino acid sequence SEQ ID No: 4 is encoded by the DNA molecule with the sequence of SEQ ID No: 8.

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