High affinity mouse anti-salmon igm monoclonal antibody, hybridoma cell strain and application thereof

Abstract

This invention relates to a high-affinity mouse anti-carp IgM monoclonal antibody, a hybridoma cell line, and their applications. The mouse anti-carp IgM monoclonal antibody is obtained from the secretion of hybridoma cell line CcM-12A12G11C9C7 with accession number CCTCC NO: C2024236. The mouse anti-carp IgM monoclonal antibody can be used for the specific detection of secreted IgM in carp using Western blot and ELISA, and can also be used for flow cytometry to identify carp IgM. + B cells. This monoclonal antibody exhibits high specificity and sensitivity, and can be used for structural analysis of carp IgM and detection of immune response levels, laying the foundation for in-depth research on the carp immune system and the establishment of a vaccine efficacy evaluation method based on antibody levels.

Description

Technical Field

[0001] This invention belongs to the field of monoclonal antibody technology, specifically relating to a high-affinity mouse anti-carp IgM monoclonal antibody, a hybridoma cell line, and their applications. Background Technology

[0002] Carp (Cyprinus carpio), belonging to the order Cypriniformes, family Cyprinidae, and genus Cyprinus, is widely loved by consumers for its tender and nutritious flesh and its auspicious connotations of good fortune and happiness in traditional Chinese culture. According to the *China Fisheries Statistical Yearbook*, my country's annual carp production has exceeded 2.8 million tons for many consecutive years, making it one of my country's important freshwater economic fish species. However, due to factors such as genetic degradation, carp diseases are frequent, causing significant economic losses to the carp farming industry. It is well known that vaccination can activate the fish's immune system and improve its resistance to specific pathogens, making it an important means of preventing fish diseases and a mainstream direction for future fish disease control. However, accurately evaluating the immunization effect of vaccines is often cumbersome, time-consuming, and lacks evaluation standards. Using monoclonal antibodies against fish immunoglobulins (Ig) as a basis, and analyzing the changes in the levels of specific antibodies (i.e., secreted Ig) in vaccinated fish to evaluate the immunization effect of vaccines, will provide a powerful tool for establishing an evaluation standard system for the immunization effect of carp vaccines.

[0003] As the earliest discovered and most abundant immunoglobulin in bony fish, IgM has been proven to play a vital role in systemic and mucosal immunity. Therefore, the development of carp IgM monoclonal antibodies will contribute to a deeper understanding of the humoral immune response patterns in carp, and will help establish methods for analyzing and detecting carp antibodies. This will, in turn, lead to the development of methods for evaluating vaccine efficacy based on carp antibody levels, and the formulation of vaccine usage protocols, thus promoting the development of carp infectious disease immunization and control technologies centered on vaccination. Summary of the Invention

[0004] This invention provides a hybridoma cell line CcM-12A12G11C9C7, which was deposited on July 16, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: C2024236.

[0005] The present invention also provides a high-affinity mouse anti-carp IgM monoclonal antibody, which is obtained by the secretion of hybridoma cells with accession number CCTCC NO: C2024236.

[0006] Furthermore, the CDR of the heavy chain variable region of the monoclonal antibody comprises the following three sequences:

[0007] HYGMN (CDRH1, SEQ ID NO.1),

[0008] WINTYTGEPTYADDFKG (CDRH2, SEQ ID NO.2),

[0009] GGDSVHYYFDV (CDRH3, SEQ ID NO.3)

[0010] The CDR of the light chain variable region of the monoclonal antibody consists of the following three sequences:

[0011] RSSQTIVHSNGNTYLE (CDRL1, SEQ ID NO.4),

[0012] RVSNRFF (CDRL2, SEQ ID NO.5),

[0013] FQGSHVPNT (CDRL3, SEQ ID NO. 6).

[0014] The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody is: QIQLVQSGPELKKPGETVKISCKASGYTFTHYGMNWVKQAPGKALKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLLINNLKNEDTATYFCARGGDSVHYYFDVWGAGTTVTVSS (SEQ ID) NO.7); Preferably, the polynucleotide encoding this amino acid is: CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACACACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGCTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGA GCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCTCATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCTAGAGGGGGGGATTCTGTCCACTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO.8);

[0015] The amino acid sequence of the variable region of the light chain of the monoclonal antibody is: DVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWFLQKPGQSPKLLIYRVSNRFFGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPNTFGGGTKLEIKRA (SEQ ID) NO.9); Preferably, the polynucleotide encoding this amino acid is: GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGACCATTGTACATAGTAATGGAAACACCTATTTAGAGTGGTTTCCTGCAGAAACCGGGCCAGTCTCCAAAGCTCCTGATCTA CAGAGTTTCCAACCGATTTTTTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGAACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCT (SEQ ID NO.10).

[0016] The present invention also provides a polynucleotide encoding the above-mentioned monoclonal antibody, or encoding the CDR region, or encoding the variable region of the heavy chain and light chain.

[0017] The present invention also provides a recombinant vector containing the polynucleotide, and a host cell containing the polynucleotide or the recombinant vector.

[0018] The present invention also provides the application of the hybridoma cell line, the monoclonal antibody, the polynucleotide, the recombinant vector, or the host cell in the preparation of a carp IgM detection kit.

[0019] Furthermore, the detection kit is an ELISA detection kit.

[0020] The present invention also provides a kit for detecting IgM in carp, the kit comprising the hybridoma cell line and / or the monoclonal antibody.

[0021] Beneficial effects: This invention uses Protein A-affinity purified carp serum natural IgM as an antigen to immunize mice and screens to obtain a hybridoma cell line CcM-12A12G11C9C7 that can specifically secrete mouse anti-carp IgM monoclonal antibody. The monoclonal antibody secreted by this hybridoma cell line CcM-12A12G11C9C7 can specifically recognize the carp-secreted form of IgM and IgM. + B cells, with their high specificity, sensitivity, and affinity, can be used for structural analysis of carp IgM and detection of immune response levels, laying the foundation for in-depth research on the carp immune system and the establishment of a vaccine efficacy evaluation method based on antibody levels. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The image shows the results of detecting purified carp serum native IgM by SDS-polyacrylamide gel electrophoresis (PAGE) under reducing and non-reducing conditions, and staining with Coomassie brilliant blue.

[0024] Figure 2 This is a graph showing the results of ELISA analysis of mouse anti-carp IgM monoclonal antibody subclasses.

[0025] Figure 3 The image shows the results of Western blot analysis of IgM bands in carp serum diluted 100 and 500 times under reducing and non-reducing conditions using mouse anti-carp IgM monoclonal antibody.

[0026] Figure 4 To detect IgM in total leukocytes of carp peripheral blood by flow cytometry + Results image of B cells.

[0027] Figure 5 To analyze mouse anti-carp IgM monoclonal antibodies for detection in carp serum via Western blot and ELISA, a Superdex gel filtration chromatography column was used. TM Graph showing the results of IgM in different elution volumes after separation using 200 Increase 10 / 300 GL (GE Healthcare).

[0028] Figure 6 This is a graph showing the binding and dissociation curves of antigen protein and antibody at different dilution concentrations when antibody affinity is determined using Bio-Layer Interferometry (BLI).

[0029] Figure 7 To verify by flow cytometry the effect of supernatants from passages 0, 5, 10, 15, and 20 of hybridoma cell line CcM-12A12G11C9C7 on IgM in total leukocytes from carp peripheral blood, the following assays were performed. + Image showing the results of B cell identification.

[0030] Figure 8 The figure shows the results of Western blot verification of the recognition of secreted IgM in carp serum by the supernatant of hybridoma cell line CcM-12A12G11C9C7 at passages 0, 5, 10, 15 and 20. Detailed Implementation

[0031] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. Unless specifically stated, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless specifically stated, the reagents and materials used in the following embodiments are commercially available.

[0032] Example 1: Preparation of a high-affinity mouse anti-carp IgM monoclonal antibody

[0033] In this embodiment, HiTrap® Protein A column (GE Healthcare) was used to affinity purify natural IgM protein from carp serum. This protein was then used as an antigen to immunize mice to obtain the hybridoma cell line CcM-12A12G11C9C7. This hybridoma cell line CcM-12A12G11C9C7 then secreted mouse anti-carp IgM monoclonal antibody. The specific process is as follows:

[0034] (1) Obtaining carp serum

[0035] Healthy carp were immersed in 30 mg / L of fish anesthetic MS-222 (Sigma). After anesthesia, they were placed in a dissecting dish with their heads covered with a towel. Scales on the lateral line of the posterior margin of the anal fin were removed. A 5 mL syringe without anticoagulant was inserted into the muscle tissue at the midline behind the anal fin until the needle reached the spine, and blood was drawn into the syringe. After the drawn blood coagulated, it was left to stand overnight at 4°C. The next day, it was centrifuged at 3000 g for 10 min at 4°C, and the supernatant serum was transferred to a clean centrifuge tube.

[0036] (2) Purification of natural IgM from carp serum

[0037] ① Equilibrate the column: Rinse the Protein A affinity purification column with 10 mL Binding buffer (1×PBS, pH=7.2).

[0038] ② Sample loading: Remove impurities by centrifuging carp serum at 4℃ and 18000 g for 30 min, then filter through a 0.22 μm filter membrane. Dilute the serum 1:3 with Binding buffer and load it onto a Protein A affinity purification column using an ÄKTA pure 25L protein purification system (GE Healthcare).

[0039] ③ Neutral washing: Rinse the Protein A affinity purification column with 20 mL Binding buffer to remove unbound contaminating proteins.

[0040] ④ Acidic elution: Rinse the Protein A affinity purification column with 0.1 M citric acid solution at pH 3.0, and collect the eluent in a centrifuge tube pre-filled with 200 μL of 1 mol / L Tris-HCl solution at pH 9.0.

[0041] ⑤ The collected eluted samples were subjected to SDS-PAGE to detect the purity of the purified carp IgM. The results are as follows: Figure 1 As shown, under reducing conditions, it exists as a heavy chain (between 70 and 100 kDa) and a light chain (between 25 and 35 kDa), while under non-reducing conditions, it exists as a monomer or polymer with a purity greater than 180 kDa, and the purity is 93%.

[0042] ⑥ The purified carp serum IgM was diluted with PBS at pH 7.4 and then ultrafiltered. Finally, the protein concentration was determined using a BCA kit (Biosharp).

[0043] (3) Mouse immunization

[0044] Five 6-8 week old BALB / c mice were used as immunogens to immunize them with the purified carp serum natural IgM from step (2). The immunization route and procedure were as follows: approximately 50 μg of carp serum natural IgM was thoroughly emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites on the back of the mice. Three booster immunizations were performed two weeks later, with each booster immunization two weeks apart. During the booster immunizations, the antigen protein was emulsified with an equal volume of Freund's incomplete adjuvant and injected subcutaneously at multiple sites on the back of the mice. On day 7 after the third booster immunization, tail vein blood was collected for ELISA to detect antibody titers.

[0045] (4) Cell fusion and culture

[0046] ① Cell fusion: Mix the spleen cells from the mice immunized in step (3) with mouse myeloma cells SP2 / 0 (ATCC) at a cell ratio of 5:1, centrifuge at 500 g for 10 min, and discard the supernatant. Take 1 mL of PEG-1450 (Sigma) and slowly add it to the test tube while shaking; then slowly add a total of 40 mL of DMEM culture medium in multiple batches. The entire process must be carried out in a 37℃ water bath, followed by centrifugation at 500 g for 10 min, removal of the supernatant, and completion of cell fusion.

[0047] ② Cell Culture: Gently break up the centrifuged cell clumps, resuspend them in HAT medium, and plate them into prepared 96-well plates containing feeder cells, 200 μL per well. Incubate at 37°C with 5% CO2. After one week, observe clonal growth under a microscope and record the number of clones per well. When the cells have grown to occupy approximately 1 / 3 of the bottom area of ​​the culture well, aspirate the culture supernatant for ELISA detection.

[0048] (5) Screening of positive clones, subcloning of hybridoma cells and mass production of monoclonal antibodies

[0049] ① Screening of positive clones: ELISA plates were coated with purified carp serum natural IgM (2 μg / mL) and incubated overnight at 4℃. The next day, after washing and blocking, 100 μL of hybridoma cell culture supernatant to be tested was added to each well, and incubated at 37℃ for 1 h. After discarding the supernatant, 100 μL of HRP-labeled goat anti-mouse IgG (Proteintech) diluted 1:5000 was added to each well, and incubated at 37℃ for 1 h. After discarding the supernatant, 100 μL of TMB chromogenic solution (Beyotime) was added to each well, and after developing at room temperature for 10–30 min, 50 μL of stop solution (2 M H2SO4) was added to each well to terminate the reaction. The absorbance was measured at 450 nm using a microplate reader. A positive result was defined as an absorbance value more than twice that of the negative control.

[0050] ② Subcloning of hybridoma cells: Hybridoma cells that tested positive by ELISA were gently washed and counted from the culture plate. The cell suspension was serially diluted with culture medium and seeded at 100 μL per well in a 96-well plate. After culturing for about 10 days, when the hybridoma cell colonies reached 1 / 3 of the area at the bottom of the well, the antibody activity in the supernatant was measured. Positive clones with antibody activity were then subjected to subcloning, for a total of 3 times. After repeated screening, the selection criteria were: high antibody titer in the cell supernatant detected by ELISA, high antibody concentration and stable secretion, Western blot detection of the monoclonal antibody recognizing carp IgM in monomeric or multimeric forms, and simultaneous use in flow cytometry to recognize carp IgM. + B cells. Table 1 shows the ELISA and flow cytometry results for different monoclonal cell lines.

[0051] Finally, a hybridoma cell line stably expressing anti-carp IgM monoclonal antibody was obtained, which was named hybridoma cell line CcM-12A12G11C9C7 and was deposited at the China Center for Type Culture Collection on July 16, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC NO: C2024236.

[0052] Table 1. Results of ELISA and flow cytometry analysis of the supernatant from the third subcloned cell line.

[0053]

[0054] ③ Large-scale production of monoclonal antibodies: Three healthy BALB / c mice around 10 weeks old were injected intraperitoneally with liquid paraffin, 0.5 mL / mouse. One week later, each pretreated mouse was injected intraperitoneally with 5 × 10⁻⁶ mol / L of paraffin. 7 Hybridoma cells were collected, and ascites was extracted from mice when their abdomens became extremely distended after 10-14 days. Extraction was repeated every 2 days, for a total of 7 mL of ascites. The extracted ascites was centrifuged at 18000 g for 10 min at 4°C. The supernatant was purified by Protein A affinity chromatography, yielding 12.4 mg of purified product, which was the mouse anti-carp IgM monoclonal antibody. This indicates that the concentration of mouse anti-carp IgM monoclonal antibody in the ascites was 1.77 mg / mL, with a titer of 1:2048000 (as shown in Table 2).

[0055] Table 2 Results of titer determination of mouse anti-carp IgM monoclonal antibody

[0056]

[0057] Note: The antibody titer is judged as follows: if the OD of the test well is greater than 0.1 and is greater than 2.1 times that of the negative control well (P / N>2.1, where P is the OD value of the test antibody at a certain dilution and N is the OD value of the negative control), it is judged as positive. The highest dilution of the antibody judged as positive is the antibody titer.

[0058] Example 2 Identification of mouse anti-carp IgM monoclonal antibody

[0059] (1) The detection process for antibody subclasses is as follows:

[0060] Purified carp serum natural IgM protein was coated onto ELISA plates and incubated overnight at 4°C. The plates were washed three times with PBST, and then 100 μL of mouse anti-carp IgM monoclonal antibody was added to each well, followed by incubation at 37°C for 2 h. After three washes, 100 μL of diluted HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA, and IgM monoclonal antibodies were added to each well, and the plates were incubated at 37°C for 1 h. After three washes, 100 μL of TMB chromogenic solution (Beyotime) was added to each well, and the plates were incubated at 37°C in the dark for 30 min. Finally, 50 μL of stop solution (2 M H2SO4) was added to each well to terminate the reaction. The absorbance was measured at 450 nm using a microplate reader. The HRP-labeled goat anti-mouse Ig subclass used in the positive reaction wells was identified as the antibody class to be tested. The identification results are as follows: Figure 2 As shown, the mouse anti-carp IgM monoclonal antibody subclass prepared in this invention is IgG1.

[0061] (2) Western blot detection, the process is as follows:

[0062] ① Sample preparation: Dilute the serum of healthy carp with PBS by 100 and 500 times respectively, add 1 / 5 volume of 5× reducing and non-reducing SDS-PAGE loading buffer (Solarbio), vortex to mix, heat in a metal bath at 100℃ for 10 min, and place on ice for later use or freeze at -80℃.

[0063] ②SDS-PAGE: Prepare 12% and 8% Tris-glycine polyacrylamide lower separating gels and 5% upper stacking gels respectively, load the samples for electrophoresis, and the electrophoresis conditions are: 80 V, 0.5 h; 120 V, 1 h.

[0064] ③ Transfer: Remove the SDS-PAGE gel from the gel plate and soak it in the prepared transfer buffer (25 mM Tris, 192 mM glycine, 15% methanol). Take a 0.22 μm PVDF membrane, cut it to a suitable size, soak it in methanol for 30 seconds to activate it, and then transfer it to the transfer buffer. Then, stack the membranes from bottom to top in the order of filter paper-PVDF membrane-gel-filter paper, ensuring that there are no air bubbles in each layer. Transfer the membrane at 300 mA semi-dry for 60 min to transfer the protein on the SDS-PAGE gel to the PVDF membrane.

[0065] ④ Blocking: After the transfer is completed, the PVDF membrane is transferred into a TBST (25 mM Tris, 150 mM NaCl, 0.1% Tween-20) solution containing 5% skim milk powder (Biosharp) and incubated at room temperature for 2 h.

[0066] ⑤ Primary antibody incubation: Discard the blocking solution, dilute the mouse anti-carp IgM monoclonal antibody to 2 μg / mL with TBST solution containing 5% skim milk powder, and incubate overnight at 4°C.

[0067] ⑥ Secondary antibody incubation: Remove the primary antibody solution, wash the membrane 3 times with TBST, each time for about 10 min; dilute the HRP-labeled goat anti-mouse IgG (H+L) antibody (Proteintech) 1:5000 with TBST solution containing 5% skim milk powder, and incubate at room temperature for 1 h.

[0068] ⑦ ECL color development: Remove the secondary antibody solution, wash the membrane 3 times with TBST, each time for about 10 min; prepare an appropriate amount of ECL color development solution (Biosharp) by mixing solution A and solution B in a 1:1 ratio, drop the color development solution onto the PVDF membrane, and after full contact, analyze and photograph it using a chemiluminescence imaging system.

[0069] (3) Flow cytometry detection, the procedure is as follows:

[0070] ① Isolation of total leukocytes from carp peripheral blood: Anesthetize healthy carp, remove scales from the lateral line of the posterior margin of the anal fin, and insert a 10 mL syringe, rinsed with the anticoagulant sodium heparin (Biosharp), into the muscle tissue at the midline behind the anal fin until the needle reaches the spine, and blood enters the syringe; add the extracted blood to DMEM medium containing sodium heparin and dilute it 7 times, then pass it through a 100 µm cell sieve to obtain a carp peripheral blood single-cell suspension; then add the single-cell suspension to the surface of 34% and 51% discontinuous Percoll (GE Healthcare) liquid, centrifuge, and aspirate the leukocytes between the two discontinuous Percoll layers to obtain the total leukocytes of the carp peripheral blood.

[0071] ② Primary antibody incubation: Wash carp peripheral blood total leukocytes twice with PBS containing 2% FBS (Yeasen), then add mouse anti-carp IgM monoclonal antibody to a final concentration of 2 μg / mL, incubate on ice for 45 min, and gently vortex the cells once every 15 min.

[0072] ③ Secondary antibody incubation: After washing the cells twice with PBS containing 2% FBS, add APC-Goat Anti-Mouse IgG (Biolegend) to a final concentration of 1 μg / mL, incubate on ice for 30 min, and gently vortex the cells once every 10 min.

[0073] ④ Flow cytometry: Wash cells twice with PBS containing 2% FBS, resuspend cells and filter, then detect IgM in total leukocytes of carp peripheral blood using a flow cytometer (BD). + The proportion of B cells.

[0074] Mouse anti-carp IgM monoclonal antibody against secreted forms of IgM and IgM in carp + The identification and specificity verification of B cells are as follows: Figures 3-4 As shown. Under reducing conditions, this monoclonal antibody recognizes the heavy chain of carp IgM, with a size between 70 and 100 kDa. Under non-reducing conditions, this monoclonal antibody can recognize both monomeric and polymeric forms of carp IgM. Furthermore, this monoclonal antibody can recognize carp IgM... + B cells were used, indicating that the monoclonal antibody can recognize the antigenic epitope on the surface of carp membrane-type IgM molecules. Sequencing revealed the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO. 7, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO. 9. The CDR regions (CDRH1, CDRH2, CDRH3) of the heavy chain variable region respectively contain the sequences shown in SEQ ID NO. 1–3, and the CDR regions (CDRL1, CDRL2, CDRL3) of the light chain variable region respectively contain the sequences shown in SEQ ID NO. 4–6.

[0075] Example 3: Detection of carp serum IgM using the monoclonal antibody ELISA provided by the present invention.

[0076] Two mL of healthy carp serum was centrifuged at 18000 g for 30 min and then filtered through a 0.22 μm filter to remove impurities. The serum sample was then loaded into a Superdex protein purification system (GE Healthcare) using PBS. TMGel filtration chromatography was performed in a 200Increase 10 / 300 GL column (GE Healthcare). Samples with an elution volume of 6-15 mL were used for both Western blot and ELISA detection. The Western blot detection method was as described in step (2) of Example 2. The ELISA detection method was as follows: (1) Dilute the samples with an elution volume of 6-15 mL by 10 times with coating buffer (0.05 M carbonate buffer, pH=9.6). Coat each well of the microplate with 100 μL of the diluted sample and incubate overnight at 4°C.

[0077] (2) After washing 5 times with PBST (10 mM PBS, 0.05% Tween-20), add 250 μL of PBST solution containing 5% skim milk powder to each well and incubate at 37°C for 2 h.

[0078] (3) After washing with PBST 5 times, the mouse anti-carp IgM monoclonal antibody was diluted to 1 μg / mL with PBST solution containing 5% skim milk powder as the primary antibody. 100 μL was added to each well and incubated at 37℃ for 1 h.

[0079] (4) After washing 5 times with PBST, dilute HRP-labeled goat anti-mouse IgG (H+L) (Proteintech) antibody with PBST solution containing 5% skim milk powder at a ratio of 1:5000 as secondary antibody, add 100 μL to each well and incubate at 37°C for 1 h.

[0080] (5) After washing with PBST 5 times, add 100 μL of TMB colorimetric solution to each well and develop the color at 37℃ in the dark for 30 min.

[0081] (6) Add 50 μL of stop solution (2 M H2SO4) to each well to stop the reaction for 10 min. After mixing, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at a wavelength of 450 nm.

[0082] Test results as follows Figure 5 As shown, the mouse anti-carp IgM monoclonal antibody can be used for the specific detection of natural IgM in carp by ELISA, with good specificity.

[0083] In addition, carp serum was serially diluted, and the highest dilution factor at which the mouse anti-carp IgM monoclonal antibody could detect carp serum was investigated by ELISA. The results are shown in Table 3, indicating that the highest dilution factor at which the mouse anti-carp IgM monoclonal antibody could detect carp serum was 51,200 times.

[0084] Table 3. ELISA validation of the highest dilution factor for detecting carp serum with mouse anti-carp IgM monoclonal antibody.

[0085]

[0086] Example 4: Determination of nucleic acid sequences of the heavy chain variable region and light chain variable region of monoclonal antibody.

[0087] Log-phase CcM-12A12G11C9C7 hybridoma cells were harvested, and total RNA was extracted using the Trizol (TAKARA) method. cDNA was generated by reverse transcription using oligo(dT)20 (Invitrogen) as primers. Then, using the cDNA as a template, the heavy chain variable region and light chain variable region genes were amplified separately using specific PCR primers. The PCR products were purified by agarose gel electrophoresis and then cloned into the pMD-18T vector via TA cloning. Sequencing and sequence analysis revealed the coding gene sequence for the heavy chain variable region as shown in SEQ ID NO. 8, encoding the protein shown in SEQ ID NO. 7; and the coding gene sequence for the light chain variable region as shown in SEQ ID NO. 10, encoding the protein shown in SEQ ID NO. 9.

[0088] Example 5: Affinity determination of mouse anti-carp IgM monoclonal antibody

[0089] This embodiment uses bio-layer interferometry (BLI) to determine the affinity of monoclonal antibodies. The specific steps are as follows:

[0090] (1) Dilute the mouse anti-carp IgM monoclonal antibody to 5 μg / mL with dilution buffer (10 mM PBS, 0.02% Tween 20, 0.2% BSA) and add 200 μL to each well of a 96-well plate;

[0091] (2) The purified carp IgM was serially diluted with dilution buffer to a final volume of 200 μL from an initial concentration of 125 nM. A total of 4 dilution gradients were performed.

[0092] (3) Install the biosensor with Protein A probe on the Gator Prime instrument and immerse it in dilution buffer for baseline measurement and record the baseline signal to eliminate background interference;

[0093] (4) Immerse the sensor in a diluted mouse anti-carp IgM monoclonal antibody solution to fix it on the sensor surface and form a biofilm layer;

[0094] (5) Immerse the sensor that forms a biofilm layer in the dilution buffer, record the new baseline signal, and record the thickness of the biofilm formed by the sensor immobilized with mouse anti-carp IgM monoclonal antibody.

[0095] (6) The sensor immobilized with carp IgM monoclonal antibody was sequentially immersed in carp IgM solutions of different dilution concentrations, and the changes in biofilm thickness caused by the binding of antigen and antibody were monitored and the binding kinetic curves were recorded.

[0096] (7) The sensor containing the complex was then immersed in the dissociation buffer for dissociation, and the dissociation kinetics curve was recorded.

[0097] (8) Binding and dissociation curves are as follows Figure 6 As shown, the data were analyzed using the Gator Prime analysis model, and the affinity constant was calculated. The results show that the affinity constant is 1.11 × 10⁻⁶. -9 M indicates that the antibody has a strong affinity.

[0098] Example 6: Analysis of hybridoma cell line passage stability and post-passage antibody secretion stability

[0099] Hybridoma cell line CcM-12A12G11C9C7 was passaged 20 times. Cells from passages 0, 5, 10, 15, and 20 were amplified and cultured, and cell supernatants were collected. Peripheral blood total leukocytes from carp were used for flow cytometry verification, and carp serum was used for Western blot verification under reducing conditions. The verification results are as follows: Figure 7 and Figure 8 As shown, the results indicated that the cell supernatants from passages 0, 5, 10, 15, and 20 had an effect on carp IgM levels. + The lack of difference in recognition of B cells and serum secreted IgM indicates that this hybridoma cell line has passage stability and post-passage antibody secretion stability.

[0100] In summary, this invention successfully prepared a mouse anti-carp IgM monoclonal antibody using hybridoma cell technology. This monoclonal antibody specifically recognizes carp IgM and can be used to detect secreted forms of IgM and IgM in carp. + B cells are of great significance for the study of the carp's immune system and the development and application of vaccines and other immunizing agents.

[0101] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A hybridoma cell line CcM-12A12G11C9C7, characterized in that, The hybridoma cell line CcM-12A12G11C9C7 has the accession number CCTCC NO: C2024236.

2. The monoclonal antibody secreted by the hybridoma cell line of claim 1.

3. The monoclonal antibody according to claim 2, characterized in that, The sequences of the heavy chain variable regions CDRH1, CDRH2, and CDRH3 of the monoclonal antibody are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively, and the sequences of the light chain variable regions CDRL1, CDRL2, and CDRL3 are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively.

4. The monoclonal antibody according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

9.

5. A polynucleotide encoding any of the monoclonal antibodies of claims 2-4.

6. A recombinant vector, characterized in that, It contains the polynucleotide of claim 5.

7. A host cell, characterized in that, It comprises the polynucleotide of claim 5 or the recombinant vector of claim 6.

8. The use of the hybridoma cell line of claim 1, the monoclonal antibody of any one of claims 2-4, the polynucleotide of claim 5, the recombinant vector of claim 6, or the host cell of claim 7 in the preparation of a carp IgM detection kit.

9. The application according to claim 8, characterized in that, The test kit is an ELISA test kit.

10. A kit for detecting IgM in carp, characterized in that, The kit contains the hybridoma cell line of claim 1 and / or any of the monoclonal antibodies of claims 2-4.

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