High affinity mouse anti-anguilla igk monoclonal antibody, hybridoma cell strain and application

Abstract

The application discloses a high-affinity mouse anti-Eudontomyzon vlk monoclonal antibody, a hybridoma cell line and application, the mouse anti-Eudontomyzon vlk monoclonal antibody can be obtained by secretion of hybridoma cell line (Hybridoma cell line) ArIgML-14D3C4D11F7, and the preservation number is CCTCC NO: C202544.The mouse anti-Eudontomyzon vlk monoclonal antibody provided by the application can be used for flow cytometry to identify IgK + B cells in eudontomyzon serum and spleen and the like, and can be used for ELISA and Western blot specific detection of eudontomyzon secreted IgK.The monoclonal antibody is specific, high in sensitivity and high in affinity, can be used for structure analysis of eudontomyzon IgK, immune response level detection and the like, lays a foundation for in-depth research of eudontomyzon immune system and establishment of a vaccine immune effect evaluation method taking an antibody level as an index.

Description

Technical Field

[0001] This invention belongs to the field of monoclonal antibody technology, specifically relating to a high-affinity mouse anti-eel Igκ monoclonal antibody, a hybridoma cell line, and its applications.

[0002] This application claims priority to the earlier application, application number 202511872227.X, entitled "A High-Affinity Mouse Anti-Eel Igλ Monoclonal Antibody, Hybridoma Cell Line and Its Application", with a priority date of December 12, 2025. Background Technology

[0003] The basic structure of immunoglobulins (Ig) consists of two heavy chains and two light chains linked by disulfide bonds. Based on the type of heavy chain, bony fish immunoglobulins can be classified into three types: IgM, IgD, and IgT. IgM was the first type of immunoglobulin discovered and is the most abundant immunoglobulin in fish, primarily distributed in the blood and playing a crucial role in systemic immunity. IgD plays a role in B cell development. IgT is a type of Ig found only in bony fish, existing as monomers in serum and as tetramers in the intestinal mucosa, primarily playing an important role in mucosal immune responses. Infection of aquatic animal pathogens often simultaneously activates both systemic and mucosal immunity. Oral and immersion vaccines can also simultaneously activate both host systemic and mucosal immunity. To comprehensively study the acquired immune system in fish and fully evaluate vaccine efficacy, simultaneous analysis of IgM is necessary. + B cells and IgT + The levels of IgM and IgT antibodies in B cells, blood, and mucus necessitate the development of antibodies against both IgM and IgT. Since the amount of natural IgT in bony fish mucus is very low, purifying natural IgT to prepare IgT antibodies is difficult. Even if IgM and IgT antibodies are successfully prepared, simultaneous detection of IgM and IgT is necessary for evaluating vaccine efficacy, which is time-consuming and labor-intensive. The light chains of bony fish immunoglobulins include three isoforms: Igκ, Igλ, and Igσ, all of which can bind to the heavy chains of IgM and IgT to form IgM and IgT. Because the constant region of the bony fish immunoglobulin heavy chain contains at least four Ig domains, while the constant region of the light chain contains only one Ig domain, fewer hybridoma cells recognize the light chain than the heavy chain during monoclonal antibody preparation, making it more difficult to obtain monoclonal antibodies against the Ig light chain. Currently, there are many monoclonal antibodies against fish Ig heavy chains, but only monoclonal antibodies against fish Ig light chains, namely those against channel catfish Igσ and Igκ, and those against tilapia Igλ.

[0004] Eels (Anguilla rostrata), belonging to the order Anguilliformes, family Anguillidae, and genus Anguilla, are one of my country's important aquaculture species. In 2023, my country's eel farming output reached 191,600 tons, accounting for 80% of the world's total output. In recent years, with the development of high-density intensive farming models, diseases caused by bacteria, viruses, and parasites have become increasingly serious, becoming one of the bottlenecks restricting the sustainable and healthy development of the eel farming industry. Research on the eel's acquired immune system and vaccine development will become the main methods for the prevention and control of eel diseases. Therefore, the development of eel Igκ monoclonal antibodies will help to gain a deeper understanding of the humoral immune response patterns in eel systemic tissues and mucous membranes, and will help to establish eel antibody analysis and detection methods. This will then lead to the establishment of methods for evaluating vaccine immunization efficacy based on eel antibody levels and the formulation of vaccine usage procedures, promoting the development of immunization and prevention technologies for eel infectious diseases centered on vaccination. Summary of the Invention

[0005] In view of this, the present invention provides a high-affinity mouse anti-eel Igκ monoclonal antibody, which is secreted by hybridoma cell line ArIgML-14D3C4D11F7 (accession number CCTCC NO: C202544).

[0006] Another objective of this invention is to provide a hybridoma cell line, ArIgML-14D3C4D11F7, which is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: C202544 and deposit date of January 7, 2025.

[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 eel Igκ, the application of which includes, but is not limited to, the preparation of an eel Igκ detection kit.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by this invention is as follows: A hybridoma cell line, ArIgML-14D3C4D11F7, was obtained by immunizing mice with natural immunoglobulin from eel serum using Protein A affinity purification as an antigen, followed by extensive screening. This hybridoma cell line exhibits stable passage and monoclonal antibody production. It was deposited on January 7, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: C202544 and classification name: Hybridoma Cell Line ArIgML-14D3C4D11F7.

[0009] This plan also includes: Monoclonal antibody secreted by hybridoma cell line ArIgML-14D3C4D11F7.

[0010] The above-mentioned mouse anti-eel Igκ monoclonal antibody and hybridoma cell line ArIgML-14D3C4D11F7 are used in the detection of eel Igκ, including the use of the above-mentioned monoclonal antibody to detect eel Igκ. + B cell sorting, or analysis of eel Igκ + The proportion of B cells, or the antibody levels used to study eels.

[0011] All of the above applications can be used to prepare relevant detection kits from hybridoma cells or monoclonal antibodies secreted by hybridoma cells.

[0012] Compared with the prior art, the beneficial effects of the present invention are: This invention uses Protein A-affinity purified natural immunoglobulins from eel serum as antigens to immunize mice and screens for a hybridoma cell line, ArIgML-14D3C4D11F7, that specifically secretes mouse anti-eel Igκ monoclonal antibodies. Furthermore, the monoclonal antibody secreted by the hybridoma cell line ArIgML-14D3C4D11F7 specifically recognizes eel secretory Igκ and Igκ. + B cells, and are highly specific, potent, responsive, and have strong affinity. Attached Figure Description

[0013] Figure 1 This is a diagram showing the results of ELISA analysis of mouse anti-eel Igκ monoclonal antibody subclasses in Example 2 of the present invention.

[0014] Figure 2 This is a graph showing the Igκ band results of eel serum diluted 100 and 500 times, respectively, by Western blot under reducing and non-reducing conditions using mouse anti-eel Igκ monoclonal antibody in Example 2 of the present invention.

[0015] Figure 3 In Example 2 of this invention, flow cytometry was used to detect Igκ in eel spleen lymphocytes. + Results image of B cells.

[0016] Figure 4 The light chain types and matching peptides identified by mass spectrometry analysis in Example 3 of this invention are shown.

[0017] Figure 5This is a graph showing the binding and dissociation curves of antibodies at different dilution concentrations with antigen proteins when surface plasmon resonance technology is used to determine antibody affinity in Example 4 of the present invention.

[0018] Figure 6 In Example 5 of this invention, flow cytometry was used to verify the hybridoma cell line ArIgML- The supernatants of passage 0, 5, 10, 15, and 20 cells from the 14D3C4D11F7 culture strain showed the effect of different levels of Igκ in eel blood. + Image showing the results of B cell identification.

[0019] Figure 7 This is a graph showing the results of Western blot verification of the recognition of secretory Igκ in eel serum by the supernatant of hybridoma cell line ArIgML-14D3C4D11F7 at passages 0, 5, 10, 15 and 20 in Example 5 of the present invention.

[0020] Figure 8 For example, in Example 6, flow cytometry was used to detect IgM in the spleen and peripheral blood leukocytes of eels. + Igκ + The proportion of cells. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1: Preparation of a high-affinity mouse anti-eel Igκ monoclonal antibody In this example, natural eel immunoglobulin was purified using Protein A affinity and used as an antigen to immunize mice to obtain the hybridoma cell line ArIgML-14D3C4D11F7. This hybridoma cell line ArIgML-14D3C4D11F7 then secreted mouse anti-eel Igκ monoclonal antibody. The specific process is as follows: (1) Purification of natural eel immunoglobulins Blood was collected from the tail vein of eels and stored overnight at 4°C. The blood was then diluted with 3000 ml of water. gCentrifuge for 10 min, collect the supernatant, and then centrifuge at 15000 rpm for 10 min to remove impurities. Filter through a 0.22 μm filter to obtain eel serum. Dilute the serum 1:4 with 1×PBS for later use. Equilibrate the Protein A affinity purification column with 1×PBS, and then co-incubate the diluted serum with the Protein A affinity purification column. Wash the Protein A affinity purification column with 20 mL of 1×PBS to remove contaminating proteins. Wash the Protein A affinity purification column with 0.1 M citric acid solution (pH=3.0), and collect the eluent in a centrifuge tube containing 200 μL of 1 M Tris-HCl solution (pH=9.0). Detect the purity of the purified eel immunoglobulin using SDS-PAGE. Dilute the purified eel immunoglobulin with 1×PBS, then concentrate by ultrafiltration, and determine the protein concentration using a BCA kit.

[0023] (2) Mouse immunization BALB / c mice were immunized with the purified natural eel immunoglobulin from step (1) for 6–8 weeks. The immunization route and procedure were as follows: approximately 50 μg of purified natural eel immunoglobulin from step (1) was 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. For each booster immunization, an equal volume of natural eel immunoglobulin was emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple sites on the back of the mice. Blood was collected from the tail on day 7 after the third booster immunization, and antibody titers were detected by ELISA.

[0024] (3) Cell fusion and culture Spleen cells from immunized mice were mixed with SP2 / 0 myeloma cells at a ratio of 5:1 in a 50 mL centrifuge tube and centrifuged at 1000 rpm for 10 min, discarding the supernatant. The centrifuge tube was placed in a 37°C water bath, and 1 mL of PEG-4000 (preheated to 37°C) was slowly added to the tube while agitating the container. Then, complete culture medium was slowly added in multiple portions, for a total of 9 mL. The tube was then centrifuged at 1000 rpm for 10 min, and the supernatant was removed, completing cell fusion.

[0025] Gently disperse the cell pellet, resuspend the cells in 2 mL of HAT medium, and seed 200 μL into each well of a prepared 96-well plate containing feeder cells. Incubate the cells at 37°C with 5% CO2 for 7 days, then 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.

[0026] (4) Screening and cloning of positive hybridoma cell lines The purified natural eel immunoglobulin (2 μg / mL) from step (1) was used to coat the ELISA plate and incubated overnight at 4°C. The next day, the plate was blocked with PBS containing 5% BSA. The primary antibody was 100 μL of cell culture supernatant from step (3), and the secondary antibody was 100 μL of HRP-labeled goat anti-mouse IgG (Biolegend, 1:2000 dilution). Finally, 100 μL of TMB chromogenic solution (Beyotime) was added to each well. After incubation 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 an ELISA reader. A positive result was defined as an absorbance value more than twice that of the negative control. The positive hybridoma cells were gently washed out of the culture plate and counted. The cell suspension was serially diluted with culture medium and seeded into 96-well plates at 100 μL per well for culture. After 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. The selection criteria were: high antibody titer, high antibody concentration and stable secretion in cell supernatant as detected by ELISA; ability to identify monomeric or multimeric forms and light chains of eel IgM as detected by Western blot; and ability to identify eel Igκ as detected by flow cytometry. + B cells. Positive clones with antibody activity were then subcloned, for a total of three times.

[0027] Table 1. Results of ELISA and flow cytometry analysis of the supernatant from the third subcloned cell line.

[0028] After repeated screening, a hybridoma cell line stably expressing anti-eel Igκ monoclonal antibody was finally obtained and named hybridoma cell line ArIgML-14D3C4D11F7. This hybridoma cell line was deposited on January 7, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: C202544 and classification name: hybridoma cell line ArIgML-14D3C4D11F7.

[0029] (5) Mass production of monoclonal antibodies Liquid paraffin was injected intraperitoneally into healthy BALB / c mice (approximately 10 weeks old). Seven to eight days later, each pretreated mouse was injected intraperitoneally with hybridoma cells (5 × 10⁻⁶ cells). 6(10-14 days later, when the mice's abdomens were extremely distended, ascites was extracted. This was repeated every two days, for a total of 10 mL of ascites. The extracted ascites was centrifuged at 4°C and 10,000 rpm / min for 10 min. The supernatant was collected and purified using a Protein A affinity purification column, yielding 8 mg of purified product, which was the mouse anti-eel Igκ monoclonal antibody. This indicated that the concentration of the mouse anti-eel Igκ monoclonal antibody in the ascites was 1 mg / mL, and the antibody titer was measured to be 1:256000.)

[0030] Table 2. Results of titer determination of a mouse anti-eel Igκ monoclonal antibody

[0031] 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 that is judged as positive is the antibody titer.

[0032] Example 2: Identification of mouse anti-eel Igκ monoclonal antibody (1) The detection process for antibody subclasses is as follows: Purified natural eel immunoglobulin (2 μg / mL) was coated onto an ELISA plate and incubated overnight at 4°C. After washing three times with PBST, the plate was blocked with PBS containing 5% BSA. The primary antibody was 100 μL of mouse anti-eel Igκ monoclonal antibody, and the secondary antibodies were 100 μL of HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM monoclonal antibodies, respectively. Finally, 100 μL of TMB chromogenic buffer (Beyotime) was added to each well, and the reaction was incubated at room temperature for 10–30 min. The reaction was then terminated by adding 50 μL of stop solution (2 M H2SO4) to each well. 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 1 As shown, the mouse anti-eel Igκ monoclonal antibody subclass prepared in this invention is IgG1.

[0033] (2) Western blot detection of antibody recognition of Ig light chain, the process is as follows: ① Sample preparation: Dilute eel serum with PBS 100 and 500 times respectively, add 1 / 4 volume of 5× reduced or non-reduced SDS-PAGE loading buffer, vortex to mix, heat in a 100℃ metal bath for 10 min, place on ice for later use or freeze at -80℃. ②SDS-PAGE: Prepare Tris-glycine polyacrylamide (12% and 8%) and 5% stacking gels. Add 5~15 μL of sample to the sample wells and perform electrophoresis at a constant voltage of 80 V. Observe the position of the protein marker. When the marker enters the separating gel, adjust the voltage to 120 V. Stop electrophoresis when the blue dye bromophenol blue reaches the bottom of the gel plate. ③ Transfer: After electrophoresis, remove the SDS-PAGE gel from the gel plate and place it in transfer buffer (192 mM M Lycine, 25 mM Tris, 15% methanol). Take a suitable PVDF membrane (0.22 μm), soak it in methanol for 30 s to activate it, and then transfer it to the transfer buffer. Stack the membranes from bottom to top in the order of filter paper-PVDF membrane-gel-filter paper, and transfer at 300 mA for 90 min to transfer the protein from the SDS-PAGE gel to the PVDF membrane.

[0034] ④ Blocking: After the transfer is completed, wash the membrane once with TBST solution (25 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH=7.5), and block at room temperature for 1 h with 5% skim milk powder (dissolved in TBST).

[0035] ⑤ Primary antibody incubation: Discard the blocking solution, add an appropriate amount of primary antibody solution (diluted with TBST solution containing 5% skim milk powder), and incubate at room temperature for 1 h.

[0036] ⑥ Secondary antibody incubation: Remove the primary antibody solution, wash the membrane 3 times with TBST for 10 min each time, add an appropriate amount of secondary antibody solution (HRP-labeled goat anti-mouse IgG antibody diluted with TBST solution containing 5% skim milk powder at a ratio of 1:5000), and incubate at room temperature for 1 h.

[0037] ⑦ ECL color development: Remove the secondary antibody solution, wash the membrane 3 times with TBST for 10 min each time, 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.

[0038] The results are as follows Figure 2 As shown, under reducing conditions, the mouse anti-eel Igκ monoclonal antibody prepared in this invention recognizes the light chain of eel Ig, which is about 25 kDa in size; under non-reducing conditions, the monoclonal antibody can recognize the monomeric or polymeric form of Ig secreted by eels.

[0039] (3) Flow cytometry detection, the procedure is as follows: ① Isolation of leukocytes from eel spleen: Anesthetize healthy eels, remove the spleen and place it in L15 culture medium, then cut it into 1 mm pieces with scissors. 3Tissue fragments were collected and then transferred to a 70 µm cell sieve. The tissue fragments were gently pressed with a 5 mL syringe plunger while L15 culture medium was slowly added during the pressing process to allow single cells to pass through the 70 µm cell sieve, thus obtaining a spleen single-cell suspension. 34% and 51% discontinuous Percoll (GE Healthcare) solutions were prepared, and the cell suspension was slowly added to the surface of the solution. The solution was centrifuged at 400 g for 30 min at 4 ℃ in a horizontal centrifuge. 3-4 mL of cells at the 34%-51% Percoll interface were collected and washed twice with PBS containing 2% FBS to obtain eel spleen leukocytes. ② Primary antibody incubation: 1×10 6 Cells were resuspended in 200 μL of PBS containing 2% FBS, and mouse anti-eel Igκ monoclonal antibody was added to a final concentration of 2 μg / mL. Cells were incubated on ice for 1 h, with gentle vortexing every 15 min. ③ Secondary antibody incubation: After washing the cells twice with PBS containing 2% FBS, add PE 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 15 min. ④ Flow cytometry: After washing the cells twice with PBS containing 2% FBS, resuspending the cells and filtering them, Igκ in eel spleen lymphocytes was detected by flow cytometry. + The proportion of B cells.

[0040] Mouse anti-eel Igκ monoclonal antibody against Igκ + B cell identification status, such as Figure 3 As shown, this monoclonal antibody can recognize eel Igκ. + B cells.

[0041] Example 3: Mass spectrometry analysis of the light chain types recognized by mouse anti-eel Igκ monoclonal antibody (1) Affinity column coupling: Add an appropriate volume of NHS activated agarose resin to an empty column, allow the resin to settle, and effluent the stock solution; wash the resin with 10-15 times the resin volume of pre-cooled 1 M hydrochloric acid, and then wash with 2-3 times the resin volume of coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3); add the prepared monoclonal antibody (dialyzed into the coupling buffer beforehand, 1-10 mg / mL) to the washed NHS activated agarose resin (NHS activated agarose resin volume: monoclonal antibody solution volume = 1: 2); couple overnight at 4°C with shaking, then collect the reaction solution and wash the medium with 3 times the resin volume of coupling buffer; add 2 times the resin volume of blocking buffer (0.1 M Tris-HCl, pH 8.5) and react with shaking at 28°C for 1 h to block all unreacted groups on the resin; effluent the blocking buffer and alternately use 10 times the resin volume of high pH washing buffer (0.1 M Tris-HCl, pH 8.5). Wash the sealed resin 3-6 times with Tris-HCl (pH 8.0~9.0) and 10 times the resin volume of low pH washing solution (0.1 M acetic acid / sodium acetate, 0.5 M NaCl, pH 4.0~5.0); then equilibrate with 10 times the resin volume of neutral equilibration solution (0.05 M Na2HPO4, pH 7.0) before use.

[0042] (2) Antigen purification and mass spectrometry identification: The column was equilibrated with 1×PBS at 10 times the resin volume, and then incubated overnight at 4°C with diluted serum (diluted with 1×PBS 1:4); the column was washed with 20 mL of 1×PBS to remove impurities; the antigen protein was eluted with 0.1 M citric acid solution (pH=3.0), and the eluent was collected in a centrifuge tube containing 200 μL of 1 M Tris-HCl solution (pH=9.0); the purity of the purified antigen was detected by SDS-PAGE; the purified antigen was identified and analyzed by mass spectrometry by Shanghai Zhongke New Life Biotechnology Co., Ltd.

[0043] Experimental results are as follows Figure 4 As shown, the results indicate that the light chain type recognized by the mouse anti-eel Igκ monoclonal antibody is Igκ.

[0044] 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) Install the COOH chip according to the OpenSPR™ instrument standard operating procedure.

[0045] (2) The detection buffer is PBST, and the initial flow rate is 150 µL / min.

[0046] (3) After the signal baseline is reached, load 200 µL of isopropanol and run for 10 s to remove air bubbles. After the baseline is reached, rinse the sample loop with PBST and remove air.

[0047] (4) After the signal reaches the baseline, adjust the flow rate to 20 µL / min.

[0048] (5) Activate the chip by loading EDC / NHS (1:1) solution.

[0049] (6) Load 200 µL of natural eel Igκ, run for 4 min, rinse the sample loop and purge with air.

[0050] (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.

[0051] (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.

[0052] (9) The results of the binding and dissociation curves are as follows: Figure 5 As shown, the affinity constant was analyzed using a One-to-One analysis model and TraceDrawer (Ridgeview Instruments ab, Sweden) software. The results showed that the affinity constant was 3.03 × 10⁻⁶. -8 M indicates that the antibody has a strong affinity.

[0053] Example 5: Stability analysis of hybridoma cell lines after passage and stability analysis of secreted antibodies after passage Hybridoma cell line ArIgML-14D3C4D11F7 was continuously passaged. Cells from passages 0, 5, 10, 15, and 20 were enlarged and cultured, and cell supernatants were collected. Western blot analysis was performed on eel serum under reducing conditions, and flow cytometry was also performed on total leukocytes from eel blood for validation. The validation results are as follows: Figure 6 and Figure 7 As shown, the results indicated that the cell supernatants from passages 0, 5, 10, 15, and 20 had an effect on serum secretory Igκ and eel Igκ levels. + The lack of difference in B cell recognition indicates that this hybridoma cell line has passage stability and post-passage antibody secretion stability.

[0054] Example 6: Analysis of IgM by mouse anti-eel Igκ monoclonal antibody + Igκ +proportion of B cells (1) Leukocytes from eel spleen and peripheral blood separated by Percoll density gradient centrifugation were aliquoted into flow cytometry tubes and the cell concentration was adjusted to 4 × 10⁻⁶ cells / mL with PBS containing 2% FBS. 6 cells / mL; (2) Aliquot the cell suspension into 1.5 mL EP tubes, 200 μL per tube, and add FITC-conjugated mouse anti-eel IgM heavy chain monoclonal antibody and APC-conjugated mouse anti-eel gκ monoclonal antibody respectively. The final antibody concentration is 2 μg / mL. Incubate on ice for 45 min, vortexing once every 15 min. (3) Wash twice with PBS containing 2% FBS and resuspend in 200 μL. Detect the cells by flow cytometry after passing through a cell filter.

[0055] Double staining results as follows Figure 8 As shown, IgM in the spleen + Igκ + B cells account for a significant portion of total IgM. + B cells account for approximately 41%, while the proportion in peripheral blood is approximately 48%; IgM in the spleen + Igκ + B cells account for Igκ + B cells account for approximately 80% of the total, with about 88% found in peripheral blood.

[0056] In summary, this invention successfully prepared a mouse anti-eel Igκ monoclonal antibody using hybridoma cell technology. This monoclonal antibody specifically recognizes eel Igκ and can be used to detect secreted forms of Igκ and Igκ in eels. + B cells are of great significance for the study of the eel's immune system and the prevention of infectious diseases.

[0057] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A hybridoma cell line ArIgML-14D3C4D11F7, characterized by: This hybridoma cell line is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: C202544 and deposit date of January 7, 2025.

2. The monoclonal antibody secreted by the hybridoma cell line ArIgML-14D3C4D11F7 as described in claim 1.

3. The use of the hybridoma cell line as described in claim 1 or the monoclonal antibody as described in claim 2 in the preparation of eel Igκ kit.

4. The application as described in claim 3, characterized in that, The kit mentioned is eel Igκ + B-cell analysis or sorting kit.

5. The application as described in claim 3, wherein the kit is an eel Igκ ELISA detection kit.

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