A mouse anti-ictalurus lebeckianus igm monoclonal antibody, hybridoma cell strain and application

Abstract

The application belongs to the field of monoclonal antibodies, and particularly relates to a mouse anti-Pelteobagrus nigro-maculatus IgM monoclonal antibody, a hybridoma cell strain secreting the monoclonal antibody and application, the CDR region of the heavy chain variable region of the mouse anti-Pelteobagrus nigro-maculatus IgM monoclonal antibody comprises three sequences as shown in SEQ ID NO. 1~3, the CDR region of the light chain variable region comprises three sequences as shown in SEQ ID NO. 4~6; the mouse anti-Pelteobagrus nigro-maculatus IgM monoclonal antibody can be obtained by the hybridoma cell strain PFM-1B7C12A1B2 with the preservation number of CCTCC NO: C2024234. The mouse anti-Pelteobagrus nigro-maculatus IgM monoclonal antibody provided by the application can be used for Western blot and ELISA specific detection of secreted IgM of Pelteobagrus nigro-maculatus, and can be used for flow cytometry identification of Pelteobagrus nigro-maculatus IgM + B cells. The monoclonal antibody has good specificity, high sensitivity and strong affinity, and can be used for structural analysis of Pelteobagrus nigro-maculatus IgM, detection of immune response level and the like, thereby laying a foundation for in-depth research on the immune system of Pelteobagrus nigro-maculatus 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 mouse anti-yellow catfish IgM monoclonal antibody, a hybridoma cell line, and its applications. Background Technology

[0002] Yellow catfish (Pelteobagrus fulvidraco), belonging to the order Siluriformes, family Bagridae, and genus Pelteobagrus, is highly favored by consumers for its delicious flavor, tender texture, and rich nutrition. According to the China Fisheries Statistical Yearbook, my country's yellow catfish production reached 622,600 tons in 2023, a 3.81% increase compared to 2022, making it one of my country's important specialty freshwater aquaculture fish. However, with the continuous deterioration of the aquaculture environment and the degradation of genetic resources, yellow catfish diseases are frequent, causing significant economic losses to the yellow catfish farming industry. The increasing use of antibiotics has led to increasingly serious problems of bacterial resistance and food safety, severely impacting the healthy and sustainable development of aquaculture. Vaccination, which can activate the fish's immune system and enhance its resistance to specific pathogens, will be an important means of preventing yellow catfish 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. Based on monoclonal antibodies against fish immunoglobulins (Ig), evaluating the immunization effect of vaccines by analyzing the changes in the levels of specific antibodies (i.e. secretory immunoglobulins) in vaccinated fish will provide a powerful tool for establishing an evaluation standard system for the immunization effect of yellow catfish 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. However, research on the yellow catfish immune system has remained stagnant due to the lack of yellow catfish IgM monoclonal antibodies as a tool. Therefore, the development of yellow catfish IgM monoclonal antibodies will contribute to a deeper understanding of the humoral immune response patterns in yellow catfish, and will help establish methods for analyzing and detecting yellow catfish antibodies. This will, in turn, lead to the development of methods for evaluating vaccine efficacy based on yellow catfish antibody levels, and the formulation of vaccine usage protocols, thus promoting the development of immunological control technologies for yellow catfish diseases centered on vaccination. Summary of the Invention

[0004] This invention provides a mouse anti-yellow catfish IgM monoclonal antibody, which is obtained by hybridoma cells secreted by a cell with accession number CCTCC NO: C2024234.

[0005] Another object of the present invention is to provide a hybridoma cell line PFM-1B7C12A1B2, the preservation number of which is CCTCC NO: C2024234.

[0006] Another objective of this invention is to provide the application of the above-mentioned monoclonal antibody or hybridoma cell line in the detection of yellow catfish IgM, the application including but not limited to the preparation of yellow catfish IgM detection kit.

[0007] To achieve the above objectives, the present invention adopts the following technical measures:

[0008] A hybridoma cell line, PFM-1B7C12A1B2, was obtained by immunizing mice with natural IgM from yellow catfish serum purified with Protein A affinity, followed by extensive screening. This hybridoma cell line was deposited on July 16, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: C2024234 and classification name: Hybridoma cell line PFM-1B7C12A1B2.

[0009] The scope of protection of this invention includes:

[0010] Monoclonal antibodies secreted by hybridoma cell line PFM-1B7C12A1B2.

[0011] The polynucleotide encoding the aforementioned monoclonal antibody.

[0012] The monoclonal antibodies described above are characterized by having a CDR region in their heavy chain variable region containing the following three amino acid sequences:

[0013] KSWMN(CDRH1, SEQ ID NO.1),

[0014] RIYPGDGDTNYNGNFKG(CDRH2, SEQ ID NO.2),

[0015] YNKDWYFDV(CDRH3, SEQ ID NO.3)

[0016] Its light chain variable region CDR region is characterized by containing the following three amino acid sequences:

[0017] KASENVVTYVS(CDRL1,SEQ ID NO.4),

[0018] GASNRYT(CDRL2, SEQ ID NO.5),

[0019] GQSYSFPLT (CDRL3, SEQ ID NO. 6).

[0020] The monoclonal antibody described above has the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO.7; preferably, the polynucleotide encoding this amino acid is shown in SEQ ID NO.8.

[0021] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.9; preferably, the polynucleotide encoding the amino acid is shown in SEQ ID NO.10.

[0022] A recombinant vector comprising the aforementioned polynucleotides.

[0023] The host cell contains the aforementioned vector or nucleic acid.

[0024] The scope of protection of this invention also includes:

[0025] The above-mentioned mouse anti-yellow catfish IgM monoclonal antibody, hybridoma cell line PFM-1B7C12A1B2, recombinant vector or host cell were used in the preparation of yellow catfish IgM detection kit.

[0026] In the above-described applications, preferably, the detection kit is an ELISA detection kit.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] This invention uses Protein A-affinity purified natural IgM from yellow catfish serum as an antigen to immunize mice and screens to obtain a hybridoma cell line, PFM-1B7C12A1B2, that can specifically secrete mouse anti-yellow catfish IgM monoclonal antibody. The monoclonal antibody secreted by this hybridoma cell line PFM-1B7C12A1B2 can specifically recognize the secreted form of IgM and membrane surface IgM from yellow catfish. + B cells, and it has good specificity, high sensitivity and strong affinity. Attached Figure Description

[0029] Figure 1 This image shows the results of SDS-polyacrylamide gel electrophoresis (PAGE) in Example 1 of the present invention, detecting purified yellow catfish serum natural IgM under reducing and non-reducing conditions and staining with Coomassie brilliant blue.

[0030] Figure 2This is a diagram showing the results of analyzing mouse anti-yellow catfish IgM monoclonal antibody subclasses using an enzyme-linked immunosorbent assay (ELISA) in Example 2 of the present invention.

[0031] Figure 3 This is a graph showing the IgM band results of yellow catfish serum diluted 100 and 500 times, respectively, detected by Western blot under reducing and non-reducing conditions using mouse anti-yellow catfish IgM monoclonal antibody in Example 2 of the present invention.

[0032] Figure 4 In Example 2 of this invention, IgM was detected in lymphocytes from the head kidney tissue of yellow catfish using flow cytometry. + Results image of B cells.

[0033] Figure 5 In Example 3 of this invention, the serum of yellow catfish was analyzed by Western blot and ELISA using mouse anti-yellow catfish IgM monoclonal antibody detection via a Superdex gel filtration chromatography column. TM Graph showing the results of IgM in different eluents after separation using 200Increase 10 / 300GL (GE Healthcare).

[0034] 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) in Example 5 of this invention.

[0035] Figure 7 In Example 6 of this invention, flow cytometry was used to verify the effect of supernatants from passages 0, 5, 10, 15, and 20 of the hybridoma cell line PFM-1B7C12A1B2 on IgM in the head and kidney tissue of yellow catfish. + Image showing the results of B cell identification.

[0036] Figure 8 This is a graph showing the results of Western blot verification of the recognition of secreted IgM in the serum of yellow catfish by the supernatant of hybridoma cell line PFM-1B7C12A1B2 at passages 0, 5, 10, 15 and 20 in Example 5 of the present invention. Detailed Implementation

[0037] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0038] Unless otherwise specified, the methods described in the following examples are conventional; the reagents and materials described are commercially available unless otherwise specified.

[0039] Example 1: Preparation of a mouse anti-yellow catfish IgM monoclonal antibody

[0040] This example uses HiTrap. TM Protein A HP (GE Healthcare) affinity-purified natural IgM protein from yellow catfish serum, and used it as an antigen to immunize mice to obtain the hybridoma cell line PFM-1B7C12A1B2. This hybridoma cell line PFM-1B7C12A1B2 then secreted mouse anti-yellow catfish IgM monoclonal antibody. The specific process is as follows:

[0041] (1) Obtaining serum from yellow catfish

[0042] Healthy yellow catfish 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. 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 3000g for 10 minutes at 4°C, and the supernatant serum was transferred to a clean centrifuge tube.

[0043] (2) Purification of natural IgM from Yellow Catfish Serum

[0044] ① Equilibrate the column: Rinse the Protein A affinity purification column with 10 mL Binding buffer (1×PBS).

[0045] ② Sample loading: Yellow catfish serum was centrifuged at 18000g for 30 minutes at 4℃ to remove impurities, then filtered through a 0.22μm filter membrane. The serum was diluted 1:3 with binding buffer and then... The protein was loaded onto a Protein A affinity purification column using a pure 25L protein purification system (GE Healthcare).

[0046] ③ Neutral washing: Rinse the Protein A affinity purification column with 20 mL Binding buffer (1×PBS) to remove unbound contaminating proteins.

[0047] ④ Acidic elution: Rinse the Protein A affinity purification column with 0.1M citric acid solution at pH 3.0, and collect the eluent in a centrifuge tube pre-filled with 200μL of 1mol / L Tris-HCl solution at pH 9.0.

[0048] ⑤ The collected eluted samples were subjected to SDS-PAGE to detect the purity of purified yellow catfish IgM. The test 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 greater than 180 kDa, and a purity of 95%.

[0049] ⑥ The purified yellow catfish serum IgM was diluted with PBS at pH 7.4 and then ultrafiltered. Finally, the protein concentration was determined using a BCA kit (Biosharp).

[0050] (3) Mouse immunization

[0051] Five 6-8 week old BALB / c mice were used as immunogens to immunize the mice with the purified yellow catfish serum natural IgM from step (2). The immunization route and procedure were as follows: approximately 50 μg of yellow catfish 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. For each booster immunization, 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.

[0052] (4) Cell fusion and culture

[0053] 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 several batches. The entire process must be carried out in a 37°C water bath. After centrifugation at 1000 rpm for 10 min, the supernatant was removed, and cell fusion was completed.

[0054] 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 and 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.

[0055] (5) Screening of positive clones, subcloning of hybridoma cells, and mass production of monoclonal antibodies.

[0056] ① Screening of positive clones: ELISA plates were coated with natural IgM (2 μg / mL) from yellow catfish serum and incubated overnight at 4°C. 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°C 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°C for 1 h. After discarding the supernatant, 100 μL of TMB chromogenic solution (Beyond) was added to each well, and the reaction was incubated at room temperature for 10–30 min. Finally, 50 μL of stop solution (2MH2SO4) 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.

[0057] 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. 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 and concentration in the cell supernatant detected by ELISA, stable antibody secretion, and the ability of the monoclonal antibody to recognize the monomeric or multimeric form of yellow catfish IgM as detected by Western blot. It could also be used for flow cytometry to identify yellow catfish IgM. + B cells, Table 1 shows the results of ELISA and flow cytometry analysis of different monoclonal cell lines.

[0058] Finally, a hybridoma cell line stably expressing anti-yellow catfish IgM monoclonal antibody was obtained, which was named hybridoma cell line PFM-1B7C12A1B2. It was deposited at the China Center for Type Culture Collection on July 16, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC NO: C2024234.

[0059] Table 1. Results of ELISA and flow cytometry analysis of the supernatant from the third subcloned cell line.

[0060]

[0061]

[0062] ③ Mass production of monoclonal antibodies: 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 g of paraffin. 7Hybridoma cells were collected, and ascites was extracted from mice when the abdomen became extremely distended after 10–14 days. Extraction was repeated every 2 days, for a total of 6 mL of ascites. The extracted ascites was centrifuged at 4°C and 10,000 rpm for 10 min. The supernatant was then purified by Protein A affinity chromatography, yielding 11 mg of purified product, which was the mouse anti-yellow catfish IgM monoclonal antibody with a concentration of 1.83 mg / mL and a titer of 1:4096000 (as shown in Table 2).

[0063] Table 2. Results of titer determination of mouse anti-yellow catfish IgM monoclonal antibody

[0064]

[0065]

[0066] 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.

[0067] Example 2: Identification of mouse anti-yellow catfish IgM monoclonal antibody

[0068] (1) The detection process for antibody subclasses is as follows:

[0069] Purified yellow catfish 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-yellow catfish IgM monoclonal antibody was added to each well, incubating 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 incubated at 37°C for 1 h. After three washes, 100 μL of TMB chromogenic solution (Beyond) was added to each well, and incubated at 37°C in the dark for 30 min. Finally, 50 μL of stop solution (2M 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-yellow catfish IgM monoclonal antibody subclass prepared in this invention is IgG2b.

[0070] (2) Western blot detection, the process is as follows:

[0071] ① Sample preparation: Dilute the serum of healthy yellow catfish 100 and 500 times with PBS respectively, then add 1 / 5 volume of 5× reducing and non-reducing SDS-PAGE loading buffer (Solarbio), vortex to mix, heat in a 100℃ metal bath for 10 min, and place on ice for later use or freeze at -80℃.

[0072] ②SDS-PAGE: Prepare 12% and 8% Tris-glycine polyacrylamide lower separating gel and 5% upper stacking gel respectively, load the samples for electrophoresis, and the electrophoresis conditions are 80V, 0.5h; 120V, 1h.

[0073] 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. Perform semi-dry transfer at 300 mA for 60 minutes to transfer the protein from the SDS-PAGE gel to the PVDF membrane.

[0074] 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 hours.

[0075] ⑤ Primary antibody incubation: Discard the blocking solution, dilute the mouse anti-yellow catfish IgM monoclonal antibody to 1 μg / mL with TBST solution containing 5% skim milk powder, and incubate overnight at 4°C.

[0076] ⑥ Secondary antibody incubation: Remove the primary antibody solution, wash the membrane 3 times with TBST for about 10 minutes each time, take HRP-labeled goat anti-mouse IgG (H+L) antibody (Proteintech) and dilute it with TBST solution containing 5% skim milk powder at a ratio of 1:5000, and incubate at room temperature for 1 hour.

[0077] 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.

[0078] (3) Flow cytometry detection, the procedure is as follows:

[0079] ① Isolation of total white blood cells from head kidney tissue of yellow catfish: Anesthetize healthy yellow catfish, cut open the body cavity through the cloaca, remove the head kidney tissue and place it in DMEM medium (Gibco), then cut it into 1mm pieces with scissors. 3 The tissue fragments were then transferred to a 100μm cell sieve (Biosharp), and the tissue fragments were gently ground with a 2mL syringe plunger. During the grinding process, DMEM culture medium was slowly added to allow single cells to pass through the 100μm cell sieve, thus obtaining a single-cell suspension of head kidney tissue. The single-cell suspension was then added to the surface of 34% and 51% discontinuous Percoll (GE Healthcare) liquids. After centrifugation, the white blood cells between the two discontinuous Percoll layers were aspirated to obtain the total white blood cells of the yellow catfish head kidney tissue.

[0080] ② Primary antibody incubation: Wash the total white blood cells of the head and kidney tissue of yellow catfish twice with PBS containing 2% FBS (Yeasen), then add mouse anti-yellow catfish IgM monoclonal antibody to a final concentration of 2 μg / mL, and incubate on ice for 45 min, gently vortexing the cells once every 15 min.

[0081] ③ Secondary antibody incubation: Wash cells twice with PBS containing 2% FBS, then add PE-Goat Anti-Mouse IgG (Biolegend) to a final concentration of 1 μg / mL, and incubate on ice for 30 min, gently vortexing the cells once every 10 min.

[0082] ④ Flow cytometry: After washing the cells twice with PBS containing 2% FBS, resuspending the cells and filtering them, IgM in the lymphocytes of the head kidney tissue of yellow catfish was detected by flow cytometry (BD). + The proportion of B cells.

[0083] Mouse anti-yellow catfish IgM monoclonal antibody against secreted IgM and IgM in yellow catfish + 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 yellow catfish IgM, which is approximately 75 kDa in size. Under non-reducing conditions, this monoclonal antibody can recognize both monomeric and polymeric forms of yellow catfish IgM. Furthermore, this monoclonal antibody can recognize yellow catfish IgM. +B cells were used, indicating that the monoclonal antibody can recognize the antigenic epitope on the surface of the yellow catfish membrane-type IgM molecule. 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 to SEQ ID NO.3, and the CDR regions (CDRL1, CDRL2, CDRL3) of the light chain variable region respectively contain the sequences shown in SEQ ID NO.4 to SEQ ID NO.6.

[0084] Example 3: Detection of serum IgM in yellow catfish using monoclonal antibody ELISA provided by the present invention.

[0085] 2 mL of healthy yellow catfish 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 filtered through PBS. Pure 25L protein purification system (GE Healthcare) loading samples into Superdex TM Gel filtration chromatography was performed on a 200Increase 10 / 300GL column (GE Healthcare). Samples with elution volumes 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:

[0086] (1) Dilute the elution volume of the sample to 6-15 mL 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.

[0087] (2) After washing 5 times with PBST (10mM PBS, 0.05% Tween-20), add 250μL of PBST solution containing 5% skim milk powder to each well and incubate at 37℃ for 2h.

[0088] (3) After washing 5 times with PBST, the mouse anti-yellow catfish 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°C for 1 h.

[0089] (4) After washing 5 times with PBST, dilute HRP-labeled goat anti-mouse IgG (H+L) antibody (Proteintech) 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.

[0090] (5) After washing with PBST 5 times, add 100 μL of TMB colorimetric solution to each well and develop the color at 37°C in the dark for 30 min.

[0091] (6) Add 50 μL of stop solution (2M H2SO4) to each well to stop the reaction for 10 min. After mixing, use an ELISA reader to detect the absorbance at a wavelength of 450 nm.

[0092] Test results as follows Figure 5 As shown, ELISA detects OD 450 The magnitude of the value corresponds to the band signal intensity detected by Western blot, indicating that the mouse anti-yellow catfish IgM monoclonal antibody can be used for ELISA to specifically detect natural IgM in yellow catfish, with good specificity.

[0093] In addition, yellow catfish serum was serially diluted, and the highest dilution factor that the mouse anti-yellow catfish IgM monoclonal antibody could detect in yellow catfish serum was investigated by ELISA. The results are shown in Table 3, indicating that the highest dilution factor that the mouse anti-yellow catfish IgM monoclonal antibody could detect in yellow catfish serum was 51,200 times, and the reaction was sensitive.

[0094] Table 3 shows the highest dilution factor of mouse anti-yellow catfish IgM monoclonal antibody that can be detected in yellow catfish serum using ELISA verification.

[0095] Yellow catfish serum dilution factor ELISA detection of OD value 50 2.130 100 2.457 200 2.658 400 2.379 800 2.168 1600 1.936 3200 1.863 6400 1.619 12800 1.336 25600 0.898 51200 0.233 102400 0.125 PBS 0.089

[0096] Example 4:

[0097] Determination of nucleic acid sequences of the heavy chain and light chain variable regions of mouse anti-yellow catfish IgM monoclonal antibody.

[0098] PFM-1B7C12A1B2 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 cDNA as a template, the heavy chain variable region and light chain variable region genes were amplified using specific PCR primers. After agarose gel electrophoresis and purification, the PCR products were cloned into the pMD-18T vector via TA cloning. Sequencing and sequence analysis were then performed to obtain the gene sequence encoding the heavy chain variable region as shown in SEQ ID NO.8, encoding the protein shown in SEQ ID NO.7; and the gene sequence encoding the light chain variable region as shown in SEQ ID NO.10, encoding the protein shown in SEQ ID NO.9.

[0099] Example 5:

[0100] Affinity assay of mouse anti-yellow catfish IgM monoclonal antibody

[0101] This embodiment uses bio-layer interferometry (BLI) to determine the affinity of monoclonal antibodies. The specific steps are as follows:

[0102] (1) Dilute the mouse anti-yellow catfish 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;

[0103] (2) The purified yellow catfish IgM was serially diluted with dilution buffer at an initial concentration of 125 nM for a total of 7 dilutions, with a final volume of 200 μL.

[0104] (3) Install the biosensor with Protein A probe on the Gator Prime instrument and immerse it in dilution buffer for baseline determination and record the baseline signal to eliminate background interference.

[0105] (4) Immerse the sensor in a diluted mouse anti-yellow catfish IgM monoclonal antibody solution to fix it on the sensor surface and form a biofilm layer;

[0106] (5) The sensor that forms the biofilm layer is then immersed in the dilution buffer, and the new baseline signal is recorded. The thickness of the biofilm formed by the sensor immobilized with mouse anti-yellow catfish IgM monoclonal antibody is also recorded.

[0107] (6) The sensor immobilized with yellow catfish IgM monoclonal antibody was sequentially immersed in yellow catfish 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.

[0108] (7) The sensor containing the complex was then immersed in the dissociation buffer for dissociation, and the dissociation kinetics curve was recorded.

[0109] (8) The results of the 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 8.42 × 10⁻⁶. -10 M indicates that the antibody has a strong affinity.

[0110] Example 6:

[0111] Analysis of hybridoma cell line passage stability and post-passage antibody secretion stability

[0112] Hybridoma cell line PFM-1B7C12A1B2 was continuously passaged to 20 generations. Cells from generations 0, 5, 10, 15, and 20 were selected for amplification and culture, and cell supernatants were collected. Total leukocytes from the head kidney of yellow catfish were used for flow cytometry verification, and yellow catfish 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, and 20 had an effect on IgM in yellow catfish. + 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.

[0113] In summary, this invention successfully prepared a mouse anti-yellow catfish IgM monoclonal antibody using hybridoma cell technology. This monoclonal antibody can specifically recognize yellow catfish IgM and can be used to detect secreted forms of IgM and IgM in yellow catfish. + B cells are of great significance for the study of the immune system of yellow catfish and the prevention of its diseases.

[0114] The above description is a preferred embodiment of the present invention and should not be construed as limiting the scope of the present invention. It should be noted that any modifications, equivalent substitutions, and improvements made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A hybridoma cell line PFM-1B7C12A1B2, the preservation number of which is CCTCC NO: C2024234.

2. The monoclonal antibody secreted by the hybridoma cell line PFM-1B7C12A1B2 according to claim 1.

3. A polynucleotide encoding the monoclonal antibody of claim 2.

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

5. The monoclonal antibody according to claim 2, wherein the amino acid sequence of the heavy chain variable region 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.

6. A recombinant vector comprising the polynucleotide of claim 3.

7. A host cell comprising the polynucleotide of claim 3 or the recombinant vector of claim 6.

8. The use of the monoclonal antibody of claim 2, the hybridoma cell line PFM-1B7C12A1B2 of claim 1, the polynucleotide of claim 3, the recombinant vector of claim 6, or the host cell of claim 7 in the preparation of a yellow catfish IgM detection kit.

9. The application according to claim 8, wherein the detection kit is an ELISA detection kit.

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