Anti-tilapia mossambica IgM monoclonal antibody, hybridoma cell strain and application thereof

Abstract

The invention discloses an anti-tilapia mossambica IgM monoclonal antibody, a hybridoma cell strain and application thereof. The CDR sequence of a heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO.1-3, and the CDR sequence of a light chain variable region of the monoclonal antibody is as shown in SEQ ID NO.4-6; the monoclonal antibody can be secreted by a hybridoma cell strain with the preservation number of CCTCC (China Center For Type Culture Collection) NO: C2023153. The monoclonal antibody provided by the invention can be used for Western blot and ELISA specific detection of tilapia mossambica secretion form IgM, can be used for flow cytometry identification of tilapia mossambica IgM + B cells, has the characteristics of good specificity, high sensitivity, strong affinity and the like, can be used for structural analysis, immune response level detection and the like of tilapia mossambica IgM, and can be used for preparing a tilapia mossambica IgM immunoassay kit. And a foundation is laid for deep research of a tilapia immune system and establishment of a tilapia vaccine immune effect evaluation method taking the antibody level as an index.

Description

Technical Field

[0001] This invention belongs to the field of monoclonal antibody technology, specifically relating to anti-tilapia IgM monoclonal antibody, hybridoma cell line and its application. Background Technology

[0002] Tilapia (Oreochromis niloticus), a globally important economic fish species, has become a key species in the global aquaculture industry due to its rapid growth, excellent meat quality, and high reproductive capacity. However, with the expansion of aquaculture scale, frequent disease outbreaks have become a major bottleneck restricting the industry's development. Among these, tilapia mortality caused by diseases such as agalactiae streptococcal disease and tilapia virus disease is particularly severe, resulting in huge economic losses for the tilapia aquaculture industry.

[0003] Currently, the control of tilapia diseases mainly relies on antibiotics. However, the long-term use of antibiotics has led to increasingly serious bacterial resistance, severely impacting the healthy and sustainable development of aquaculture. Against this backdrop, vaccination, as a sustainable disease prevention method, can activate the fish's immune system and enhance its resistance to specific pathogens. It will be a crucial means of preventing tilapia diseases and a mainstream direction for future fish disease control. However, accurately evaluating the effectiveness of vaccines is often cumbersome, time-consuming, and lacks standardized evaluation criteria. Using monoclonal antibodies based on fish immunoglobulins (Ig) to analyze the changes in the levels of specific antibodies (essentially Ig) in vaccinated fish to evaluate vaccine efficacy will provide a powerful tool for establishing a standardized evaluation system for tilapia vaccine efficacy.

[0004] As the earliest discovered and most abundant immunoglobulin in bony fish, IgM has been proven to play a vital role in humoral immunity, particularly in systemic and mucosal immunity. Therefore, the development of monoclonal antibodies against tilapia IgM will contribute to a deeper understanding of the humoral immune response patterns in tilapia and will help establish methods for analyzing and detecting tilapia antibodies. This will, in turn, lead to the development of methods for evaluating vaccine efficacy based on tilapia antibody levels and the formulation of vaccine usage protocols, ultimately promoting the development of tilapia disease prevention and control technologies centered on vaccination. Summary of the Invention

[0005] This invention provides a high-affinity mouse anti-tilapia IgM monoclonal antibody, which is secreted by hybridoma cells with accession number CCTCC NO: C2023153.

[0006] Another object of the present invention is to provide a hybridoma cell line OnM-14G10B1E1, the preservation number of which is CCTCC NO: C2023153.

[0007] Another objective of this invention is to provide the application of the above-mentioned monoclonal antibody or hybridoma cell line in the detection of tilapia IgM, including but not limited to the preparation of tilapia IgM detection kits.

[0008] To achieve the above objectives, the present invention adopts the following technical measures: The first aspect of this invention provides an anti-tilapia IgM monoclonal antibody, wherein the sequences of the heavy chain variable regions CDRH1, CDRH2, and CDRH3 of the monoclonal antibody are shown in SEQ ID NO. 1-3, and the sequences of the light chain variable regions CDRL1, CDRL2, and CDRL3 of the monoclonal antibody are shown in SEQ ID NO. 4-6, respectively. Specifically: CDRH1: DYSMH (SEQ ID NO.1); CDRH2: WINTETGEPTYADDFKG (SEQ ID NO.2); CDRH3: YGRRPGYFAV (SEQ ID NO.3); CDRL1: KASQDVVTAVA (SEQ ID NO.4); CDRL2: WASTRHT (SEQ ID NO.5); CDRL3: QQYSSYPFT (SEQ ID NO. 6).

[0009] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0010] The amino acid sequence of the heavy chain variable region: QIQLVQSGPELKKPGETVKISCKASDYTFTDYSMHWVKQAPGKGLKWMAWINTETGEPTYADDFKGRFAFSFEISASASTAYLQINNLKNEDTATYFCAGYGRPRGYFAVWGAGTTVTVSS (SEQ ID NO.7) The amino acid sequence of the light chain variable region: DIVMTQSHKFMSTSVGDRVSITCKASQDVVTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSESGTDFTLTISNVQSEDLADYFCQQYSSYPFTFGSGTKLEIR (SEQ ID NO.8) A second aspect of the invention provides a polynucleotide encoding the above-described monoclonal antibody.

[0011] Further, the aforementioned nucleic acid molecule includes polynucleotide A (as shown in SEQ ID NO. 9) encoding the heavy chain variable region of a mouse anti-tilapia IgM monoclonal antibody and polynucleotide B (as shown in SEQ ID NO. 10) encoding the light chain variable region of the mouse anti-tilapia IgM monoclonal antibody. Preferably, when the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 7, the nucleotide sequence of nucleic acid molecule A can be: CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGATTATAACCTTCACAGACTATTCAATGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGCCTGGATAAACACTGAGACTGGTGAGCCAACATAT GCAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTTGAAATCTCTGCCAGTACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCTGGCTACGGTAGGCCTCGGGGGTATTTCGCTGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO.9) When the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.8, the nucleotide sequence of nucleic acid molecule B can be: GACATTTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGTATCACCTGCAAGGCCAGTCAGGATGTGGTTACTGCTGTAGCCTGGTATCAACAGAAACCAGGTCAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGG CACACTGGAGTCCCTGATCGCTTCACAGGCAGTGAATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAGCAGTTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAGATAAGA (SEQ ID NO.10) A third aspect of the present invention provides a recombinant vector comprising the aforementioned polynucleotides.

[0012] A fourth aspect of the invention provides a host cell comprising the aforementioned polynucleotide or recombinant vector.

[0013] The fifth aspect of the present invention provides a hybridoma cell line OnM-14G10B1E1, which was deposited on June 1, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: C2023153.

[0014] Furthermore, the hybridoma cell line OnM-14G10B1E1 or its passaged cell line can secrete the aforementioned anti-tilapia IgM monoclonal antibody.

[0015] Furthermore, the above-mentioned anti-tilapia IgM monoclonal antibody was obtained by injecting the hybridoma cell line OnM-14G10B1E1 into mice and then collecting the ascites fluid from the mice, followed by separation and purification.

[0016] The sixth aspect of the present invention provides the application of the above-mentioned mouse anti-tilapia IgM monoclonal antibody, polynucleotide, recombinant vector, host cell or hybridoma cell line OnM-14G10B1E1 in the preparation of a tilapia IgM detection kit.

[0017] Preferably, the detection kit includes: a colloidal gold immunoassay kit, a chemiluminescence kit, an enzyme-linked immunosorbent assay kit, or an immunofluorescence kit.

[0018] A seventh aspect of the present invention provides a kit for detecting IgM in tilapia, the kit comprising the above-mentioned mouse anti-tilapia IgM monoclonal antibody, polynucleotide, recombinant vector, host cell or hybridoma cell line OnM-14G10B1E1.

[0019] Beneficial effects: This invention uses Protein A affinity-purified tilapia serum natural IgM as an antigen to immunize mice and screens for a hybridoma cell line, OnM-14G10B1E1, that can specifically secrete monoclonal antibodies against tilapia IgM. This hybridoma cell line has the significant characteristics of stable passage and stable antibody secretion. Moreover, the monoclonal antibody secreted by the hybridoma cell line OnM-14G10B1E1 can specifically recognize the secreted form of IgM and IgM from tilapia. + B cells, with a titer of 1:2048000 and an affinity constant of up to 6.02 × 10⁻⁶. -9 M, characterized by high specificity, high potency, rapid response, and strong affinity, helps to elucidate the patterns of humoral immune responses in tilapia after pathogen infection, lays the foundation for the development of novel vaccines and immune enhancers, provides a reliable tool for tilapia immunological research, and is of great significance for promoting the healthy development of tilapia aquaculture. Attached Figure Description

[0020] 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.

[0021] Figure 1 This is a diagram showing the results of SDS-polyacrylamide gel electrophoresis (PAGE) in Example 1 of the present invention, detecting purified tilapia serum natural IgM under reducing and non-reducing conditions and staining with Coomassie brilliant blue.

[0022] Figure 2 This is a graph showing the results of ELISA analysis of mouse anti-tilapia IgM monoclonal antibody subclasses in Example 2 of the present invention.

[0023] Figure 3 This is a graph showing the IgM band results of tilapia serum diluted 100 and 500 times, respectively, detected by Western blot under reducing and non-reducing conditions using mouse anti-tilapia IgM monoclonal antibody in Example 2 of the present invention.

[0024] Figure 4In Example 2 of this invention, flow cytometry was used to detect IgM in peripheral blood lymphocytes of tilapia. + Results image of B cells.

[0025] Figure 5 In Example 3 of this invention, the detection of tilapia serum by ELISA using mouse anti-tilapia IgM monoclonal antibody was performed via a Superdex gel filtration chromatography column. TM Graph showing the results of IgM in different elution volumes after separation using 200 Increase 10 / 300 GL (GE Healthcare).

[0026] Figure 6 This is a graph showing the binding and dissociation curves of antibodies at different dilutions with antigen proteins when surface plasmon resonance technology (SPR) is used to determine antibody affinity in Example 4 of this invention.

[0027] Figure 7 In Example 5 of this invention, flow cytometry was used to verify the effect of cell supernatants from passages 0, 5, 10, 15, 20, and 25 of the hybridoma cell line OnM-14G10B1E1 on IgM levels in tilapia blood. + Image showing the results of B cell identification.

[0028] Figure 8 This is a graph showing the results of Western blot verification of the recognition of secreted IgM in tilapia serum by the supernatant of hybridoma cell line OnM-14G10B1E1 at passages 0, 5, 10, 15, 20 and 25. Detailed Implementation

[0029] 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 the present invention are conventional reagents, methods, and equipment in this technical field; unless specifically stated, the reagents and materials used in the following embodiments are all commercially available.

[0030] Example 1: Preparation of mouse anti-tilapia IgM monoclonal antibody This embodiment uses HiTrap. ®Protein A column (GE Healthcare) used affinity purification of natural IgM protein from tilapia serum. Mice were then immunized with this protein as an antigen, and a hybridoma cell line, OnM-14G10B1E1, was obtained via cell fusion technology. This OnM-14G10B1E1 cell line then secreted mouse anti-tilapia IgM monoclonal antibody. The specific process is as follows: (1) Obtaining tilapia serum Healthy tilapia 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 behind the anal fin were removed. A 10 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.

[0031] (2) Purification of natural IgM from tilapia serum ① Equilibrate the column: Rinse the Protein A affinity purification column with 10 mL Binding buffer (1 × PBS).

[0032] ② Sample loading: Remove impurities by centrifuging tilapia 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).

[0033] ③ Neutral washing: Rinse the Protein A affinity purification column with 20 mL Binding buffer to remove unbound contaminating proteins.

[0034] ④ Acidic elution: Elute 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.

[0035] ⑤ The collected eluted samples were subjected to SDS-PAGE to detect the purity of purified tilapia IgM. The detection results are as follows: Figure 1 As shown, under reducing conditions, it exists as a heavy chain (approximately 75 kDa) and a light chain (approximately 25 kDa), while under non-reducing conditions, it exists as a monomer or polymer with a purity of approximately 95%, and as a monomer or polymer with a purity greater than 180 kDa.

[0036] ⑥ The purified tilapia serum IgM was diluted with PBS at pH 7.4 and then ultrafiltered. Finally, the protein concentration was determined using a BCA kit (Biosharp).

[0037] (3) Mouse immunization Five 6-8 week old BALB / c mice were used as immunogens to immunize them with the purified tilapia serum natural IgM from step (2). The immunization route and procedure were as follows: approximately 50 μg of tilapia 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 blood was collected for ELISA to detect antibody titers.

[0038] (4) Cell fusion and culture ① Cell fusion: Mouse spleen cells and mouse myeloma cells SP2 / 0 were mixed at a ratio of 5:1, centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. 1 mL of PEG-4000 (Sigma) was slowly added to the test tube while agitating; then, a total of 9 mL of complete culture medium was slowly added in multiple additions. The entire process must be carried out in a 37℃ water bath. After centrifugation at 1000 rpm for 10 min, the supernatant was removed, and cell fusion was completed.

[0039] ② Cell Culture: Gently break up the centrifuged cell clumps, resuspend them in HAT medium, and plate them into 200 μL of prepared feeder cells in 96-well plates. Incubate at 37°C with 5% CO2. After one week, observe clonal growth under a microscope and record the number of clones in each well. When the cells have grown to cover approximately 1 / 3 of the bottom area of ​​the culture well, aspirate the culture supernatant for ELISA detection.

[0040] (5) Screening of positive clones, subcloning of hybridoma cells and mass production of monoclonal antibodies ① Screening of positive clones: ELISA plates were coated with tilapia 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 the reaction was stopped after 10–30 min at room temperature by adding 50 μL of stop solution (2 M H2SO4) to each well. 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.

[0041] 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 about 10 days of culture, 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. Subcloning was performed on positive clones with antibody activity, for a total of 3 times. After repeated screening, the selection criteria were: high antibody titer in the supernatant, high antibody concentration and stable secretion; the monoclonal antibody could recognize tilapia IgM in both monomeric and multimeric forms, and could also recognize tilapia IgM. + B cells. The results of ELISA and flow cytometry analysis of different monoclonal cell lines are shown in Table 1.

[0042] Table 1. Results of ELISA and flow cytometry analysis of the third subcloned cell line. Finally, a hybridoma cell line stably expressing anti-tilapia IgM monoclonal antibody was obtained, which was named hybridoma cell line OnM-14G10B1E1. This cell line was deposited at the China Center for Type Culture Collection on June 1, 2023, at Wuhan University, Wuhan, China, with accession number CCTCCNO: C2023153.

[0043] ③ Mass production of monoclonal antibodies: Healthy BALB / c mice around 10 weeks old were intraperitoneally injected with liquid paraffin, 0.5 mL / mouse. One week later, each pretreated mouse was intraperitoneally injected 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 6.5 mL of ascites. The extracted ascites was centrifuged at 17000 g for 10 min at 4°C. The supernatant was purified by Protein A affinity chromatography, yielding 11.9 mg of purified product, which was the mouse anti-tilapia IgM monoclonal antibody, indicating a concentration of 1.83 mg / mL in the ascites. The concentration of the obtained mouse anti-tilapia IgM monoclonal antibody was adjusted to 1 mg / mL, first diluted 1000-fold and then serially diluted 2-fold. The antibody titer was then determined by ELISA. The results are shown in Table 2, indicating a titer of 1:2048000 for the mouse anti-tilapia IgM monoclonal antibody.

[0044] Table 2. Results of titer assay for mouse anti-tilapia IgM monoclonal antibody 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.

[0045] Example 2 Identification of mouse anti-tilapia IgM monoclonal antibody (1) The detection process for antibody subclasses is as follows: Purified tilapia serum native 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-tilapia 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 the antibody class to be tested.

[0046] The results of the identification are as follows Figure 2 As shown, the mouse anti-tilapia IgM monoclonal antibody subclass prepared in this invention is IgG2b.

[0047] (2) Western blot detection, the process is as follows: ① Sample preparation: Dilute the serum of healthy tilapia 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℃.

[0048] ②SDS-PAGE: Prepare 12% and 8% Tris-glycine polyacrylamide lower separating gels and 5% upper stacking gels, respectively, and load them for electrophoresis. The electrophoresis conditions are 80 V for 0.5 h and 120 V for 1 h.

[0049] 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. Stack the layers 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 at 300 mA semi-dry for 60 min to transfer the protein from the SDS-PAGE gel to the PVDF membrane.

[0050] 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.

[0051] ⑤ Primary antibody incubation: Discard the blocking solution, dilute the mouse anti-tilapia IgM monoclonal antibody to 2 μg / mL with TBST solution containing 5% skim milk powder, and incubate overnight at 4°C.

[0052] ⑥ Secondary antibody incubation: Remove the primary antibody solution, wash the membrane 3 times with TBST for about 10 min each time, take HRP-labeled goat anti-mouse IgG (H+L) antibody and dilute it with TBST solution containing 5% skim milk powder at a ratio of 1:5000, and incubate at room temperature for 1 h.

[0053] ⑦ ECL color development: Remove the secondary antibody solution, wash the membrane 3 times with TBST, each time for about 10 minutes, 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.

[0054] (3) Flow cytometry detection, the procedure is as follows: ① Isolation of total leukocytes from peripheral blood of tilapia: Anesthetize healthy tilapia and collect peripheral blood from the caudal peduncle using a 5 mL syringe containing heparin sodium (Sigma). Dilute the blood 7 times with DMEM medium (Gibco) and add it to the surface of 34% and 51% discontinuous Percoll (GE Healthcare) liquid. After gradient centrifugation, aspirate the leukocytes between the two discontinuous Percoll layers to obtain the total leukocytes from the peripheral blood of tilapia.

[0055] ② Primary antibody incubation: After washing tilapia peripheral blood total leukocytes twice with PBS containing 2% FBS (Yeasen), mouse anti-tilapia IgM monoclonal antibody was added to a final concentration of 2 μg / mL. The cells were incubated on ice for 45 min, with the cells gently swirled once every 15 min.

[0056] 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.

[0057] ④ Flow cytometry: After washing the cells twice with PBS containing 2% FBS, resuspending the cells and filtering them, IgM in tilapia peripheral blood lymphocytes was detected by flow cytometry (BD). + The proportion of B cells.

[0058] Mouse anti-tilapia IgM monoclonal antibody against secreted forms of IgM and IgM in tilapia + The identification and specificity verification of B cells are as follows: Figures 3-4 As shown. Figure 3 As shown, under reducing conditions, this monoclonal antibody specifically recognizes the heavy chain of tilapia IgM, with a size of approximately 75 kDa. Under non-reducing conditions, this monoclonal antibody can recognize both monomeric and polymeric forms of tilapia IgM, and even after the serum is diluted 500-fold, a clear IgM band can still be detected under both reducing and non-reducing conditions, indicating that this monoclonal antibody has high affinity and detection sensitivity. Furthermore, as... Figure 4 As shown, this monoclonal antibody can recognize tilapia IgM. + B cells indicate that the recognition epitope of this monoclonal antibody is located on the surface of the tilapia membrane-type IgM molecule.

[0059] Example 3: ELISA detection of serum IgM in tilapia Serum from healthy tilapia was passed through a molecular sieve. Samples with elution volumes of 6-15 mL were collected for ELISA detection. The detection steps are as follows: (1) Dilute the samples with elution positions of 6-15 mL 10 times with coating buffer (0.05 M carbonate buffer, pH = 9.6) and add them to the microplate for coating, 100 μL per well, and incubate overnight at 4℃.

[0060] (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.

[0061] (3) After washing 5 times with PBST, the mouse anti-tilapia IgM monoclonal antibody was diluted to 2 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°C for 1 h.

[0062] (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 1:2000 as secondary antibody, add 100 μL to each well and incubate at 37°C for 1 h.

[0063] (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.

[0064] (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.

[0065] Test results as follows Figure 5 As shown, the mouse anti-tilapia IgM monoclonal antibody can be used for the specific detection of natural tilapia IgM by ELISA, with good specificity.

[0066] In addition, tilapia serum was serially diluted, and the highest dilution factor at which the mouse anti-tilapia IgM monoclonal antibody could detect tilapia serum was investigated by ELISA. The results are shown in Table 3, indicating that the highest dilution factor at which the mouse anti-tilapia IgM monoclonal antibody could detect tilapia serum was 51,200 times.

[0067] Table 3. ELISA validation of the highest dilution factor of mouse anti-tilapia IgM monoclonal antibody that can detect tilapia serum. Example 4: Monoclonal Antibody Affinity Assay This embodiment uses surface plasmon resonance (SPR) technology to determine antibody affinity. The specific steps are as follows: (1) According to OpenSPR TM Install the COOH chip according to the instrument's standard operating procedure.

[0068] (2) Start running at the maximum flow rate (150 µL / min) with PBST as the detection buffer.

[0069] (3) After the signal baseline is reached, load 200 µL of IPA (isopropanol), run for 10 s to remove air bubbles, and after the baseline is reached, rinse the sample loop with buffer and remove air.

[0070] (4) After the signal reaches the baseline, adjust the PBST flow rate to 20 µL / min.

[0071] (5) Activate the chip by loading EDC / NHS (1:1) solution.

[0072] (6) Load the sample with 200 µL of Protein A affinity-purified tilapia IgM diluted with PBST for 4 min, rinse the sample loop and purge with air.

[0073] (7) Load 200 µL of Blocking solution, rinse the sample loop with PBST and purge with air, and observe the baseline for 5 min to ensure stability.

[0074] (8) Dilute the antibody to be tested with PBS to 80 nM, 40 nM, 20 nM, 10 nM, 5 nM and 2.5 nM respectively, and load them at 20 µL / min. The binding time between antigen and antibody is 4 min and the natural dissociation time is 6 min.

[0075] (9) The results of the binding and dissociation curves are as follows: Figure 6 As shown, using a One-to-One analysis model and TraceDrawer (Ridgeview Instruments ab, Sweden) software, affinity constant analysis was performed. The results showed that the affinity constant of this mouse anti-tilapia IgM monoclonal antibody reached 6.02 × 10⁻⁶. -9 M indicates that the antibody has a strong affinity.

[0076] Example 5: Analysis of the stability of hybridoma cell lines after passage and the stability of secreted antibodies after passage. Hybridoma cell line OnM-14G10B1E1 was continuously passaged to 25 generations. Cells from generations 0, 5, 10, 15, 20, and 25 were selected for amplification culture, and cell supernatants were collected. Total leukocytes from tilapia were used for flow cytometry verification, and tilapia 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 cell supernatants from generations 0, 5, 10, 15, 20, and 25 had an effect on tilapia 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.

[0077] Example 6: Determination of nucleic acid sequences of the heavy chain variable region and light chain variable region of monoclonal antibody. OnM-14G10B1E1 hybridoma cells in logarithmic growth phase were used. Total RNA was extracted using the Trizol (TAKARA) method, and 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 using specific PCR primers. The PCR products were purified by agarose gel electrophoresis and cloned into the pMD-18T vector via TA cloning. Sequencing and sequence analysis were then performed to obtain the coding gene for the heavy chain variable region as shown in SEQ ID NO.9, encoding the protein shown in SEQ ID NO.7; the coding gene for the light chain variable region is shown in SEQ ID NO.10, encoding the protein shown in SEQ ID NO.8. The CDR regions (CDRH1, CDRH2, CDRH3) of the heavy chain variable region contain the sequences shown in SEQ ID NO.1-3, respectively, and the CDR regions (CDRL1, CDRL2, CDRL3) of the light chain variable region contain the sequences shown in SEQ ID NO.4-6, respectively.

[0078] In summary, this invention successfully screened a hybridoma cell line, OnM-14G10B1E1, that specifically secretes mouse anti-tilapia IgM using hybridoma cell technology. The line is stable in passage and produces stable antibody secretion. Furthermore, a mouse anti-tilapia IgM monoclonal antibody secreted by OnM-14G10B1E1 was prepared from this cell line. This antibody specifically recognizes IgM and IgM secreted by tilapia. + B cells, characterized by high specificity, high titer, high responsiveness, and strong affinity, are of great significance for the study of the tilapia immune system and the evaluation of immunoprophylactic techniques for diseases.

[0079] 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 monoclonal antibody against tilapia IgM, 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.1-3, respectively, and the sequences of the light chain variable regions CDRL1, CDRL2, and CDRL3 of the monoclonal antibody are shown in SEQ ID NO.4-6, respectively.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. A polynucleotide encoding the monoclonal antibody of any one of claims 1-2.

4. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide of claim 3.

5. A host cell, characterized in that, The host cell contains the polynucleotide of claim 3 or the recombinant vector of claim 4.

6. A hybridoma cell line OnM-14G10B1E1, characterized in that, The hybridoma cell line OnM-14G10B1E1 has the accession number CCTCC NO: C2023153.

7. The use of the monoclonal antibody of any one of claims 1-2, the polynucleotide of claim 3, the recombinant vector of claim 4, the host cell of claim 5, or the hybridoma cell line of claim 6 in the preparation of a tilapia IgM detection kit.

8. The application according to claim 7, characterized in that, The detection kits include: colloidal gold immunoassay kits, chemiluminescence kits, enzyme-linked immunosorbent assay kits, or immunofluorescence kits.

9. A kit for detecting IgM in tilapia, characterized in that, The kit contains any of the monoclonal antibodies of claims 1-2 or the hybridoma cell line of claim 6.

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