Mouse anti-Channa argus IgM monoclonal antibody, hybridoma cell line and application thereof

Abstract

The present invention belongs to the technical field of monoclonal antibody, and specifically relates to a murine anti-channa argus IgM monoclonal antibody, a hybridoma cell line and their applications. The CDR regions of the heavy chain variable region of the murine anti-channa argus IgM monoclonal antibody contain three sequences shown in SEQ ID NO.1-3, and the CDR regions of the light chain variable region contain three sequences shown in SEQ ID NO.4-6; it is secreted by the hybridoma cell line with the preservation number of CCTCC NO: C2023234. The murine anti-channa argus IgM monoclonal antibody provided by the present invention can be used for specific detection of secreted IgM of channa argus by Western blot and ELISA, and can be used for flow cytometry to identify channa argus IgM<supgt;+< / supgt> B cells. This monoclonal antibody has good specificity, high sensitivity and strong affinity, laying a foundation for the in-depth study of the immune system of channa argus and the establishment of a vaccine immune effect evaluation method with antibody level as an index.

Description

Technical Field

[0001] The invention belongs to the technical field of monoclonal antibodies, and specifically relates to a high-affinity mouse anti-Channa argus IgM monoclonal antibody, a hybridoma cell line and applications. Background Art

[0002] Channa argus belongs to the order Perciformes, family Liomorpha, genus Liomorpha. It is also known as black fish, mullet, money fish, black Litou, etc. It is favored by consumers for its delicious meat, tender meat and rich nutrition. According to the China Fisheries Statistical Yearbook, the annual output of Channa argus in my country has exceeded 1.56 million tons for many consecutive years, and it has become one of the important freshwater farmed fish in my country. However, with the continuous deterioration of the aquaculture water environment and the degradation of germplasm, Channa argus diseases are frequent, among which the death of Channa argus caused by infectious diseases such as bacterial and viral diseases is very serious, causing huge economic losses to the Channa argus farming industry. Due to the continuous use of antibiotics, the problem of bacterial resistance is becoming increasingly serious, seriously affecting the healthy and sustainable development of the aquaculture industry. Vaccination can activate the fish immune system and improve the body's resistance to specific pathogens. It is an important means to prevent diseases of Channa argus and is also the mainstream direction of fish disease prevention and control in the future. However, the accurate evaluation of the immune effect of vaccines is often cumbersome, time-consuming, and lacks evaluation standards. The immune effect of vaccines can be evaluated by analyzing the changes in the antibody titers (antigen-specific Ig secreted into body fluids) in the blood and mucus of vaccinated fish based on monoclonal antibodies to fish immunoglobulins (Ig). This will provide a powerful tool for establishing an evaluation standard system for the immune effect of snakehead fish vaccines.

[0003] As the earliest and most abundant immunoglobulin found in teleost fish, IgM has been shown to play a very important role in the systemic and mucosal immunity of fish. Therefore, the development of IgM monoclonal antibodies for Channa argus will help us gain a deeper understanding of the humoral immune response of Channa argus, and help establish the analysis and detection methods for Channa argus antibodies, and then establish a method for evaluating the immune effect of vaccines based on Channa argus antibody levels and formulate vaccine use regulations, thus promoting the development of immune prevention and control technology for Channa argus infectious diseases with vaccination as the core. Summary of the invention

[0004] The invention provides a mouse anti-Channa argus IgM monoclonal antibody. The monoclonal antibody is secreted by a hybridoma cell with a deposit number of CCTCCNO: C2023234.

[0005] Another object of the present invention is to provide a hybridoma cell line CaM-11D9B4B9, the deposit number of the hybridoma cell line is CCTCC NO: C2023234.

[0006] Another object of the present invention is to provide the use of the above monoclonal antibody or hybridoma cell line in detecting Channa argus IgM, and the use includes but is not limited to the preparation of a Channa argus IgM detection kit.

[0007] In order to achieve the above object, the present invention adopts the following technical measures:

[0008] A hybridoma cell line CaM-11D9B4B9, which was obtained by immunizing mice with Protein A affinity-purified natural IgM from snakehead serum after a large number of screening. The hybridoma cell line has stable passage and stable monoclonal antibody production. The hybridoma cell was deposited in the China Center for Type Culture Collection on August 1, 2023, address: Wuhan University, Wuhan, China, with a collection number of CCTCC NO: C2023234, and a classification name: Hybridoma cell line CaM-11D9B4B9.

[0009] The protection scope of the present invention includes:

[0010] Monoclonal antibody secreted by hybridoma cell line CaM-11D9B4B9.

[0011] A polynucleotide encoding the above monoclonal antibody.

[0012] The CDR region of the heavy chain variable region of the above-mentioned monoclonal antibody is characterized by comprising the following three sequences:

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

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

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

[0016] The CDR region of the light chain variable region is characterized by comprising the following three sequences:

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

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

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

[0020] The amino acid sequence of the heavy chain variable region of the monoclonal antibody described above is shown in SEQ ID NO.7; preferably, the polynucleotide encoding the amino acid is shown in SEQ ID NO.9;

[0021] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.8; preferably, the polynucleotide encoding the amino acid is shown in SEQ ID NO.10.

[0022] A nucleic acid molecule, which is a polynucleotide encoding the above-mentioned monoclonal antibody, or encoding the CDR region, or encoding the heavy chain and light chain variable regions;

[0023] A recombinant vector comprising the above polynucleotide;

[0024] A host cell comprising the above-mentioned vector or nucleic acid.

[0025] The protection scope of the present invention also includes:

[0026] Application of the mouse anti-Channa argus IgM monoclonal antibody, hybridoma cell line CaM-11D9B4B9, nucleic acid molecule, recombinant vector or host cell in the preparation of a Channa argus IgM detection kit.

[0027] In the above application, preferably, the detection kit is an ELISA detection kit.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention uses Protein A affinity purified natural IgM from Channa argus serum as an antigen to immunize mice and screen a hybridoma cell line CaM-11D9B4B9 that can specifically secrete mouse anti-Channa argus IgM monoclonal antibody. The hybridoma cell line has the remarkable characteristics of stable passage and stable secretion of antibodies. Moreover, the monoclonal antibody secreted by the hybridoma cell line CaM-11D9B4B9 can specifically recognize the secreted form of IgM and IgM + B cells have good specificity, high titer, sensitive response and strong affinity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure is the result of detecting the purified natural IgM in the serum of Channa argus under reducing conditions (Reducing) and non-reducing conditions (Non-reducing) by SDS-polyacrylamide gel electrophoresis (PAGE) in Example 1 of the present invention and staining with Coomassie Brilliant Blue.

[0031] Figure 2 This is a graph showing the results of analyzing mouse anti-Channa argus IgM monoclonal antibody subclasses by ELISA in Example 2 of the present invention.

[0032] Figure 3 This is a graph showing the results of Western blot in Example 2 of the present invention, in which Ig M bands of Channa argus serum diluted 100 and 500 times respectively were detected using mouse anti-Channa argus IgM monoclonal antibody under reducing and non-reducing conditions.

[0033] Figure 4 Detection of IgM in spleen tissue of Channa argus by flow cytometry in Example 2 of the present invention + Results for B cells.

[0034] Figure 5 In Example 3 of the present invention, the mouse anti-Channa argus IgM monoclonal antibody was analyzed by ELISA to detect the Channa argus serum through a gel filtration column Superdex TM Graph showing the results of IgM in different elution volumes after separation using 200Increase 10 / 300GL (GE Healthcare).

[0035] Figure 6 This is a graph showing the binding and dissociation curves of antibodies at different dilution concentrations and antigen proteins when the antibody affinity is determined using surface plasmon resonance technology (SPR) in Example 4 of the present invention.

[0036] Figure 7 The flow cytometry method was used to verify the effect of the supernatant of the 0th, 5th, 10th, 15th, 20th and 25th generations of the hybridoma cell line CaM-11D9B4B9 on the IgM in the spleen tissue of Channa argus. + B cell recognition results diagram.

[0037] Figure 8 This is a graph showing the results of using Western blot to verify the recognition of secretory IgM in Channa argus serum by the supernatants of the 0th, 5th, 10th, 15th, 20th and 25th generations of hybridoma cell line CaM-11D9B4B9 in Example 5 of the present invention. DETAILED DESCRIPTION

[0038] The technical scheme 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 part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

[0040] Example 1: Preparation of a high-affinity mouse anti-Channa argus IgM monoclonal antibody

[0041] This example uses Protein A column (GE Healthcare) was used to affinity purify the natural IgM protein from the serum of Channa argus, and it was used as an antigen to immunize mice to obtain the hybridoma cell line CaM-11D9B4B9, which was then secreted by the hybridoma cell line CaM-11D9B4B9 to produce mouse anti-Channa argus IgM monoclonal antibody. The specific process is as follows:

[0042] (1) Obtaining Channa argus serum

[0043] Healthy snakehead fish were immersed in 30 mg / L fish anesthetic MS-222 (Sigma), and after anesthesia, they were placed in a dissecting dish, and their heads were covered with a towel. The scales on the lateral line at the rear edge of the anal fin were removed, and 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 entered the syringe. After the drawn blood coagulated, it was placed at 4°C overnight and centrifuged at 4°C and 3000g for 10 min the next day. The upper serum was aspirated and transferred to a clean centrifuge tube.

[0044] (2) Purification of natural IgM from Channa argus serum

[0045] ① Equilibrate the column: rinse the Protein A affinity purification column with 10 mL Binding buffer (1×PBS);

[0046] ② Loading: The snakehead serum was centrifuged at 4°C and 15,000 rpm for 30 min to remove impurities, then filtered through a 0.22 μm filter membrane, and diluted 1:3 with Binding buffer and filtered. pure 25L protein purification system (GE Healthcare) was loaded onto a Protein A affinity purification column;

[0047] ③ Neutral washing: Wash the Protein A affinity purification column with 20 mL Binding buffer (1×PBS) to remove unbound proteins;

[0048] ④ Acid elution: Rinse the Protein A affinity purification column with 0.1 M citric acid solution at pH = 3.0, and collect the eluate in a centrifuge tube pre-filled with 200 μL of 1 mol / L Tris-HCl solution at pH = 9.0;

[0049] ⑤ Take the collected elution samples and perform SDS-PAGE to detect the purity of the purified Channa argus IgM. The test results are as follows: Figure 1As shown, under reducing conditions, it is a heavy chain (about 75 kDa) and a light chain (about 25 kDa), and under non-reducing conditions, it is a monomer or polymer form greater than 180 kDa, with a purity of 93%;

[0050] ⑥ The purified snakehead serum IgM was diluted with PBS at pH 7.4 and then ultrafiltered, and the protein concentration was determined using a BCA kit (Biosharp).

[0051] (3) Mouse immunization

[0052] Take 5 BALB / c mice aged 6 to 8 weeks, and use the purified natural IgM from Channa argus serum in step (2) as an immunogen to immunize the mice. The immunization route and procedure are as follows: about 50 μg of natural IgM from Channa argus serum is fully mixed and emulsified with an equal volume of Freund's complete adjuvant, and injected subcutaneously at multiple points on the back of the mouse. Two weeks later, three booster immunizations are performed, each with an interval of two weeks. During the booster immunization, the antigen protein is mixed and emulsified with an equal volume of Freund's incomplete adjuvant, and injected subcutaneously at multiple points on the back. On the 7th day after the third booster immunization, tail blood is collected for ELISA to detect antibody titer.

[0053] (4) Cell fusion and culture.

[0054] ① Cell fusion: Mix mouse spleen cells and mouse myeloma cells SP2 / 0 at a cell ratio of 5:1, centrifuge at 1000rpm for 10min, and discard the supernatant. Pipette 1mL PEG-4000 (Sigma) and slowly add it to the test tube while shaking; then slowly add a total of 9mL complete culture medium several times. The entire process must be carried out in a 37°C water bath, then centrifuge at 1000rpm for 10min, remove the supernatant, and complete cell fusion;

[0055] ②Cell culture: Gently flick the cell clumps after centrifugation, resuspend them in HAT medium, and spread them in the prepared 96-well plate of feeder cells, 200 μL per well. Place at 37°C, 5% CO 2 After one week, observe the clone growth under a microscope and record the number of clones in each well. When the cells grow to occupy about 1 / 3 of the bottom of the culture well, aspirate the culture supernatant for ELISA detection.

[0056] (5) Screening of positive clones, subcloning of hybridoma cells, and mass production of monoclonal antibodies

[0057] ① Screening of positive clones: ELISA plates were coated with natural IgM (2 μg / mL) from snakehead serum and incubated at 4°C overnight. The next day, 100 μL of the culture supernatant of the hybridoma cells to be tested was added to each well after washing and blocking, 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. The supernatant was discarded, and 100 μL of TMB colorimetric solution (Biyuntian) was added to each well. After color development at room temperature for 10 to 30 minutes, 50 μL of stop solution (2MH 2 SO 4 ) to terminate the reaction. Use an enzyme-labeled instrument to detect the absorbance at a wavelength of 450 nm. If the absorbance is more than 2 times that of the negative control, it is considered positive;

[0058] ② Subcloning of hybridoma cells: Gently elute and count the hybridoma cells that are positive in ELISA detection from the culture plate, and continuously dilute the cell suspension with culture medium and inoculate 100 μL per well in a 96-well plate. After culturing for about 10 days, when the hybridoma cell colonies have grown to 1 / 3 of the area at the bottom of the well, begin to measure the antibody activity in the supernatant. Subclone the positive clones with antibody activity again, and perform a total of 3 times. After repeated screening, the screening conditions are: ELISA detection of high antibody titer, high antibody concentration and stable secretion in the cell supernatant, Western blot detection of the monoclonal antibody can recognize the monomer or polymer form of Channa argus IgM, and can also be used for flow cytometry to identify Channa argus IgM + B cells, Table 1 shows the results of ELISA and flow cytometry of different monoclonal cell lines.

[0059] Finally, a hybridoma cell line that stably secretes anti-Channa argus IgM monoclonal antibodies was obtained, which was named hybridoma cell line CaM-11D9B4B9. The hybridoma cell was deposited in the China Center for Type Culture Collection on August 1, 2023, address: Wuhan University, Wuhan, China, with the collection number CCTCCNO: C2023234, and the classification name: Hybridoma cell line (Hybridoma cell line) CaM-11D9B4B9.

[0060] Table 1 ELISA and flow cytometry results of the supernatant of the third subcloned cell line

[0061]

[0062]

[0063] ③Mass production of monoclonal antibodies: 10-week-old healthy BALB / c mice were intraperitoneally injected with liquid paraffin (0.5 mL / mouse). One week later, 5 × 10 6hybridoma cells, and extract ascites when the mouse abdomen is extremely swollen after 10 to 14 days, and extract once every 2 days, and extract a total of 9.5 mL of ascites. The extracted ascites was centrifuged at 4°C and 10000 rpm / min for 10 minutes, and the supernatant was purified by affinity chromatography to obtain a total of 16.7 mg of purified product, namely the mouse anti-Channa argus IgM monoclonal antibody, indicating that the concentration of the mouse anti-Channa argus IgM monoclonal antibody in the ascites was 1.75 mg / mL, and the titer was 1:4096000 (as shown in Table 2).

[0064] Table 2 Results of the titer determination of mouse anti-Channa argus IgM monoclonal antibody

[0065]

[0066]

[0067] Note: The basis for judging the antibody titer is: if the OD of the test well is greater than 0.1 and 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 measured at a certain dilution multiple, and N is the OD value of the negative control well), it is judged as positive, and the highest dilution multiple of the antibody judged as positive is the antibody titer.

[0068] Example 2: Identification of mouse anti-Channa argus IgM monoclonal antibody

[0069] (1) Detection of antibody subclasses: The process is as follows:

[0070] Purified natural IgM protein from Channa argus serum 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-Channa argus IgM monoclonal antibody was added to each well and incubated at 37°C for 2 h. After washing three times, diluted HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA and IgM monoclonal antibodies were added to each well, 100 μL, and incubated at 37°C for 1 h. After washing three times, 100 μL of TMB colorimetric solution (Biyuntian) was added to each well and incubated at 37°C in the dark for 30 min. Then 50 μL of stop solution (2M H 2 SO 4 ) to terminate the reaction. The absorbance was detected at 450 nm using an enzyme marker, and the HRP-labeled goat anti-mouse Ig subclass used in the positive reaction well was used as the antibody type to be tested. Figure 2 As shown, it is shown that the subclass of the mouse anti-Channa argus IgM monoclonal antibody prepared by the present invention is IgG1.

[0071] (2) Western blot detection, the process is as follows:

[0072] ① Sample preparation: Serum of healthy Channa argus was diluted 100 and 500 times with PBS, respectively, and 1 / 5 volume of 5× reduced and non-reduced SDS-PAGE loading buffer (Solarbio) was added, respectively, vortexed and mixed, heated in a 100℃ metal bath for 10 min, and placed on ice for later use or frozen at -80℃;

[0073] ②SDS-PAGE: Prepare 12% and 8% Tris-glycine polyacrylamide lower separation gel and 5% upper concentration gel respectively, load the samples for electrophoresis, the electrophoresis conditions are 80V, 0.5h; 120V, 1h;

[0074] ③ Transfer: Remove the SDS-PAGE gel from the gel plate, soak it in the prepared transfer buffer (25mMTris, 192mM Glycine, 15% methanol), take a 0.22μm PVDF membrane, cut it to a suitable size, soak it in methanol for 30s to activate it, and then transfer it to the transfer buffer. Stack it from bottom to top in the order of filter paper-PVDF membrane-gel-filter paper, making sure there are no bubbles in each layer, transfer the protein on the SDS-PAGE gel to the PVDF membrane at 300mA for 60min;

[0075] ④ Blocking: After the transfer, the PVDF membrane was transferred into TBST (25 mMTris, 150 mM NaCl, 0.1% Tween-20) solution containing 5% skim milk powder (Biosharp) and incubated at room temperature for 2 h;

[0076] ⑤ Primary antibody incubation: discard the blocking solution, dilute the mouse anti-Channa argus IgM monoclonal antibody to 1 μg / mL with TBST solution containing 5% skim milk powder, and incubate at 4°C overnight;

[0077] ⑥ Secondary antibody incubation: remove the primary antibody solution, wash the membrane with TBST for 3 times, each time for about 10 minutes, 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 hour;

[0078] ⑦ECL color development: remove the secondary antibody solution, wash the membrane three 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, add the color development solution dropwise onto the PVDF membrane, and use a chemiluminescence imager to analyze and take pictures after full contact.

[0079] (3) Flow cytometry detection, the process is as follows:

[0080] ① Isolation of total leukocytes from spleen tissue of Channa argus: Anesthetize healthy Channa argus, cut open the body cavity of Channa argus from the cloaca, remove spleen tissue and place it in DMEM culture medium, and cut it into 1 mm pieces with scissors. 3The tissue fragments were then moved to a 100 μm cell sieve, and the tissue fragments were gently rubbed with a 2 mL syringe piston. During the pressing process, DMEM culture medium was slowly added to allow single cells to pass through the 100 μm cell sieve to obtain a single cell suspension of spleen tissue; the single cell suspension was then added to the liquid surface of 34% and 51% discontinuous Percoll (GE Healthcare), and the white blood cells between the two layers of discontinuous Percoll were aspirated after centrifugation to obtain the total white blood cells of the spleen tissue of Channa argus;

[0081] ② Primary antibody incubation: Wash the total leukocytes of Channa argus spleen tissue twice with PBS containing 2% FBS (Yeasen), then add mouse anti-Channa argus IgM monoclonal antibody at a final concentration of 2 μg / mL, incubate on ice for 45 minutes, and gently vortex the cells once every 15 minutes;

[0082] ③ Secondary antibody incubation: Wash the cells twice with PBS containing 2% FBS, then add APC-Goat Anti-Mouse IgG (Biolegend) at a final concentration of 1 μg / mL, incubate on ice for 30 min, and gently vortex the cells once every 10 min;

[0083] ④ Flow cytometry: After washing the cells twice with PBS containing 2% FBS, resuspend the cells and filter them, and then use flow cytometry (BD) to detect IgM in the spleen tissue of Channa argus. + The proportion of B cells.

[0084] Response of mouse anti-Chanaria argus IgM monoclonal antibody to secretory IgM and IgM + B cell recognition and specificity verification Figures 3 - 4 As shown. Under reducing conditions, the monoclonal antibody recognizes the heavy chain of the secretory form of IgM of Channa argus, which is about 75kDa in size. Under non-reducing conditions, the monoclonal antibody can recognize the monomer or polymer form of the secretory form of IgM of Channa argus. In addition, the monoclonal antibody can recognize Channa argus IgM + B cells, indicating that the monoclonal antibody can recognize the antigenic epitope on the surface of the membrane-type IgM molecule of Channa argus. After sequencing, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8, wherein the CDR region of the heavy chain variable region includes the sequence shown in SEQ ID NO.1 to SEQ ID NO.3, and the CDR region of the light chain variable region includes the sequence shown in SEQ ID NO.4 to SEQ ID NO.6.

[0085] Example 3: Detection of serum IgM in Channa argus using the monoclonal antibody ELISA provided by the present invention

[0086] Take healthy snakehead serum and pass it through molecular sieves, and take samples with elution volumes of 6-14 mL for ELISA testing. The testing steps are as follows:

[0087] (1) Dilute the sample with an elution volume of 6-14 mL with coating buffer (0.05 M carbonate buffer, pH = 9.6), coat each well of the ELISA plate with 100 μL of the diluted sample, and incubate at 4°C overnight;

[0088] (2) After washing five times with PBST (10 mM PBS, 0.05% Tween-20), 250 μL of PBST solution containing 5% skim milk powder was added to each well and incubated at 37°C for 2 h;

[0089] (4) After washing with PBST for 5 times, mouse anti-Channa argus IgM monoclonal antibody was diluted to 1 μg / mL in 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;

[0090] (5) After washing with PBST five times, HRP-labeled goat anti-mouse IgG (H+L) (Proteintech) antibody was diluted at a ratio of 1:2000 with PBST solution containing 5% skim milk powder as the secondary antibody, 100 μL was added to each well, and incubated at 37°C for 1 h;

[0091] (6) After washing with PBST five times, 100 μL of TMB colorimetric solution was added to each well and color was developed at 37°C in the dark for 30 min;

[0092] (7) Add 50 μL of stop solution (2M H 2 SO 4 ) The reaction was terminated for 10 min, and the absorbance was detected using an enzyme reader at a wavelength of 450 nm after mixing.

[0093] Test results such as Figure 5 As shown, it is shown that mouse anti-Channa argus IgM monoclonal antibody can be used for ELISA to specifically detect natural IgM of Channa argus with good specificity.

[0094] In addition, the serum of Channa argus was diluted in multiples, and the highest dilution multiple of Channa argus serum that could be detected by the mouse anti-Channa argus IgM monoclonal antibody was explored by ELISA experiment. The test results are shown in Table 3, indicating that the highest dilution multiple of Channa argus serum that could be detected by the mouse anti-Channa argus IgM monoclonal antibody was 25600 times.

[0095] Table 3 ELISA validation of the highest dilution of Channa argus serum detected by mouse anti-Channa argus IgM monoclonal antibody

[0096]

[0097]

[0098] Example 4: Monoclonal Antibody Affinity Determination

[0099] This example uses surface plasmon resonance technology (SPR) to measure antibody affinity. The specific steps are as follows:

[0100] (1) Install the COOH chip according to the OpenSPRTM instrument standard operating procedure;

[0101] (2) Start running at maximum flow rate (150 μL / min) with PBST as the detection buffer;

[0102] (3) After reaching the signal baseline, load 200 μL of IPA (isopropanol) and run for 10 s to eliminate bubbles. After reaching the baseline, rinse the sample loop with buffer and evacuate it with air.

[0103] (4) After the signal reached the baseline, adjust the PBST flow rate to 20 μL / min;

[0104] (5) Load EDC / NHS (1:1) solution to activate the chip;

[0105] (6) Load 200 μL of Protein A affinity-purified Channa argus IgM diluted in PBST and run for 4 min, rinse the sample loop, and empty it with air;

[0106] (7) Load 200 μL of Blocking solution, rinse the sample loop with PBST, and evacuate it with air. Observe the baseline for 5 minutes to ensure stability.

[0107] (8) The antibody to be detected was diluted with PBS to 80 nm, 40 nm, 20 nm, 10 nm, 5 nm and 2.5 nm, respectively, and loaded at 20 μL / min. The binding time of antigen and antibody was 4 min; the natural dissociation time was 6 min;

[0108] (9) The results of the binding and dissociation curves are as follows Figure 6 As shown, the affinity constant was analyzed using the One To One analysis model and TraceDrawer (Ridgeview Instruments ab, Sweden) software. The results showed that the affinity constant was 2.11×10 -8 M, indicating that the antibody has a strong affinity.

[0109] Example 5: Analysis of the stability of hybridoma cell lines and secreted antibodies after subculture

[0110] The hybridoma cell line CaM-11D9B4B9 was continuously subcultured to the 25th generation, and the cells of the 0th, 5th, 10th, 15th, 20th and 25th generations were selected for amplification and culture, and the cell supernatant was collected. The total leukocytes of the spleen of Channa argus were taken for flow cytometry verification and the serum of Channa argus was taken for Western blot verification under reducing conditions. The verification results are shown in Figure 7 and Figure 8 As shown, the results showed that the cell supernatants of passages 0, 5, 10, 15, 20 and 25 had an effect on the IgM + There was no difference in the recognition of secretory IgM in serum by B cells, indicating that the hybridoma cell line had the stability of passage and the stability of secreted antibodies after passage.

[0111] In summary, the present invention successfully prepared mouse anti-Channa argus IgM monoclonal antibody by hybridoma cell technology, which can specifically recognize Channa argus IgM and can be used to detect Channa argus secretory IgM and IgM + B cells are of great significance for the research of the immune system and vaccine development of Channa argus.

[0112] The above description is a preferred embodiment of the present invention, which cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A hybridoma cell line CaM-11D9B4B9, the deposit number of which is CCTCC NO: C2023234.

2. The monoclonal antibody secreted by the hybridoma cell line CaM-11D9B4B9 according to claim 1.

3. A polynucleotide encoding the monoclonal antibody according to claim 2.

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

8. A recombinant vector comprising the polynucleotide according to claim 3.

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

8. Use of the IgM monoclonal antibody according to claim 2, the hybridoma cell line CaM-11D9B4B9 according to claim 1, the polynucleotide according to claim 3, the recombinant vector according to claim 6 or the host cell according to claim 7 in the preparation of a Channa argus IgM detection kit.

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

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