Monoclonal antibody specifically combined with clostridium perfringens beta2 toxin recombinant protein, hybridoma cell strain and application

Abstract

The invention belongs to the technical field of biology, and discloses a monoclonal antibody specifically combined with clostridium perfringens (C.p) beta 2 toxin recombinant protein, a hybridoma cell strain and application. The invention provides a monoclonal antibody which is specifically combined with beta 2 toxin protein and a hybridoma cell strain CPB2-2A4 which secretes the monoclonal antibody and has the preservation number of CCTCC NO: C2024312, the monoclonal antibody only reacts with the C.p beta 2 toxin protein and does not have cross reaction with other pathogens which infect cattle and sheep, and the specificity is extremely high; according to the kit, a competitive ELISA technology is utilized, a mouse monoclonal antibody specifically combined with C.p beta2 toxin recombinant protein is taken as a competitive antibody, C.p beta2 toxin antibodies in serum of various animal sources can be detected, and species limitation of sample sources is avoided. In addition, the kit is good in repeatability. The kit has low technical requirements during operation, can be widely popularized and used in production, and is applied to C.p epidemiological investigation and vaccine immune level monitoring.

Description

Technical Field

[0001] The present invention relates to a monoclonal antibody specifically binding to Clostridium perfringens (Cp) β2 toxin recombinant protein, a hybridoma cell line and application, belonging to the field of biotechnology. Background Art

[0002] Clostridium perfringens ( Clostridium perfringens , Cp) is an obligate anaerobic Gram-positive bacterium that is part of the normal gastrointestinal flora of humans and animals. When the host's internal environment changes, it can cause a variety of diseases in humans and animals, such as gas gangrene and human food poisoning, piglet diarrhea, calf enterotoxemia, lamb dysentery, poultry necrotic enteritis, etc. The main pathogenic factors of this bacterium are more than 20 exotoxins and hydrolases such as α, β, ε and ι secreted by it. The mature toxins are absorbed by the intestinal mucosa and spread to various tissues and organs such as the kidneys, lungs, liver and brain through the blood circulation. It can cause enterotoxemia in animals (goats, sheep and less common cattle), quickly kill animals and cause huge economic losses. Therefore, the prevention and control of this disease is of great significance.

[0003] The β2 toxin of Clostridium perfringens is encoded by the cpb2 gene, and all seven toxin types (AG types) of Clostridium perfringens can produce β2 toxin. In 1997, Gibert first identified the β2 toxin of Clostridium perfringens, which has an isoelectric point (pI) of 5.4 to 5.5 and is lethal to mice. Although this toxin is called β2 toxin, its amino acid sequence identity with β toxin is only 15%, and it has no obvious homology with other identified Clostridium perfringens toxin genes. A structurally complete Cp β2 toxin contains 265 amino acids, with a molecular size of approximately 31 kDa, and contains a signal peptide composed of 30 amino acids, which will be removed during the transcription and processing of β2 toxin. Therefore, the mature β2 toxin protein is a peptide chain composed of 235 amino acids, with a molecular weight of 27.67 kDa and an isoelectric point of 5.01. β2 toxin has the activity of perforating toxins, which can damage the cell membranes of various host cells. It can also affect the distribution of calcium ions in nerve cell membranes, thereby affecting the function of nerve tissue and destroying normal nerve conduction. It plays an important role in gastrointestinal diseases of animals. For example, strains of Clostridium perfringens that produce Beta2 toxin can cause necrotic enteritis in piglets and enterocolitis in horses. Therefore, in the development of a new generation of genetically engineered vaccines for Clostridium perfringens, the role of β2 toxin protein is also very important. Vaccine immunization is the most effective means to prevent Clostridium perfringens disease. The use of serological detection methods to evaluate the antibody levels of various toxin proteins of Clostridium perfringens in the serum of vaccinated animals after vaccination is extremely important for testing the quality of Clostridium perfringens vaccines and developing high-quality vaccines. However, there is currently no commercial ELISA kit for detecting β2 toxin antibodies of Clostridium perfringens in China. In previous studies, our laboratory established an indirect ELISA method for detecting Cp β2 toxin antibodies in sheep serum. This method has strong specificity and high sensitivity, and can be used to determine the level of Cp β2 toxin antibodies in serum. However, the false positive rate of indirect ELISA is relatively high, and it only detects the antibody level in the serum of specific animals. Therefore, it is necessary to establish a method that can detect the level of Cp β2 toxin antibodies in the serum of multiple animals.

[0004] Monoclonal antibody technology is to fuse a single B lymphocyte that produces antibodies with a bone marrow tumor cell to obtain a hybrid cell that can produce antibodies and proliferate indefinitely, and thereby produce monoclonal antibodies with high specificity. Competitive ELISA is a method for qualitative or quantitative detection of antigens or antibodies. Its principle is based on the competitive binding of antigens and antibodies. Therefore, the purity requirements for samples are relatively low, and detection can be performed even in complex sample matrices. Competitive ELISA is simple to operate, low in cost, and suitable for the detection of a large number of serum samples from a variety of animals, making it suitable for large-scale screening. The preparation of monoclonal antibodies to the β2 toxin protein of Clostridium perfringens, and the use of the monoclonal antibodies to develop a competitive ELISA kit for the detection of antibodies to the β2 toxin of Clostridium perfringens, will have important application value for the prevention and treatment of Clostridium perfringens disease. Summary of the invention

[0005] The purpose of the present invention is to provide a hybridoma cell line CPB2-2A4 which can secrete a monoclonal antibody that specifically binds to a Clostridium perfringens β2 recombinant protein.

[0006] Another object of the present invention is to provide a monoclonal antibody that specifically binds to the recombinant protein of Clostridium perfringens β2 toxin.

[0007] Another object of the present invention is to provide a competitive ELISA kit for detecting Clostridium perfringens β2 toxin antibodies in serum.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is: The present invention provides a hybridoma cell line CPB2-2A4 of a monoclonal antibody that specifically binds to a recombinant protein of Clostridium perfringens β2, which was deposited in the China Center for Type Culture Collection on October 16, 2024, at Wuhan University, Wuhan, China; the deposit number is CCTCC NO: C2024312, and the classification name is: Hybridoma cell line (Hybridoma cellline) CPB2-2A4.

[0009] The present invention provides a monoclonal antibody that specifically binds to a recombinant protein of Capsella perfringens β2 toxin. The heavy chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2.

[0010] Heavy chain amino acid sequence (SEQ ID NO.1): QIQLVQSGPELKKPGETVKFSCKASGYTFTNYGMNWMKQAPGKGLKWMGWINTYTGEATYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARDYYGSTYGDFEYWGQGTTLTVSS Light chain amino acid sequence (SEQ ID NO.2): DIVLTQSPASLAVSLGQRATISCRASQSVSASTFSSIHWYQQKPGQPPKLLIKYASDLKSGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWQIPYTFGGGTKLAIKRA The heavy chain nucleotide sequence encoding the monoclonal antibody is shown in SEQ ID NO.3, and the light chain nucleotide sequence encoding the monoclonal antibody is shown in SEQ ID NO.4.

[0011] Heavy chain nucleotide sequence (SEQ ID NO.3): CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGTTCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGAACTGGATGAAACAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGGCAACATATGC TGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGAGATTACTACGGTAGTACCTACGGAGACTTTGAATACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGC Light chain nucleotide sequence (SEQ ID NO.4): GACATTTGTGCTGACTCAGTCTCCTGCTTCCTTAGCTGTTTCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGCCAAAGTGTCAGTGCATCTACCTTTAGTTCTATACACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCAAGTATGCATCCG ACCTCAAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATACTGCAACATATTACTGTCAGCACAGTTGGCAGATTCCCTATACGTTCGGAGGGGGGACCAAGCTTGGCGATAAAACGGGCT The monoclonal antibody that specifically binds to the Cp β2 toxin recombinant protein is produced by the hybridoma cell line with the deposit number CCTCC NO: C2024312. The monoclonal antibody specifically binds to the Cp β2 toxin recombinant protein and competitively binds to the Cp β2 toxin recombinant protein with Cp positive serum. The preparation method of the monoclonal antibody that specifically binds to the Cp β2 toxin recombinant protein is as follows: the hybridoma cell line CCTCC NO: C2024312 is intraperitoneally injected into BALB / c female mice, the ascites of the BALB / c female mice is obtained, centrifuged, the middle layer of pale yellow ascites is collected, and the monoclonal antibody that specifically binds to the Cp β2 toxin recombinant protein is obtained after purification.

[0012] The present invention provides the use of a monoclonal antibody in preparing a reagent or a kit for detecting Clostridium perfringens Cp β2 toxin antibodies in serum. The present invention provides a competitive ELISA kit for detecting Cp β2 toxin antibodies in serum. The competitive ELISA kit for detecting Cp β2 toxin antibodies in serum is prepared by using Cp β2 toxin recombinant protein as antigen and secreting a monoclonal antibody that specifically binds to the Cp β2 toxin recombinant protein as a competitive antibody.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a monoclonal antibody specifically binding to Cp β2 toxin protein and a hybridoma cell line CPB2-2A4 secreting the monoclonal antibody. The monoclonal antibody secreted by the CCTCC NO: C2024312 cell line only reacts with the Cp β2 toxin protein, and does not cross-react with other pathogens that infect cattle and sheep, and has extremely high specificity; the kit uses competitive ELISA technology, and uses a mouse monoclonal antibody that specifically binds to the Cp β2 toxin recombinant protein as a competitive antibody, which can detect serum antibodies from a variety of animal sources, avoiding species restrictions on sample sources. In addition, the kit has good repeatability. The technical requirements for the operation of the kit are relatively loose, and it can be widely promoted and used in production, and applied to Cp epidemiological surveys and vaccine immunity level monitoring.

[0014] (1) The present invention uses soluble expressed Cp β2 toxin recombinant protein as an antigen to immunize mice, and then takes mouse spleen cells and fuses them with myeloma SP2 / 0 cells to screen a hybridoma cell line CPB2A4 that secretes specific monoclonal antibodies, and its deposit number is CCTCC NO: C2024312. The present invention inoculates the CCTCC NO: C2024312 cell line into the peritoneal cavity of mice, collects the mouse ascites to purify antibodies, and obtains a purified monoclonal antibody that specifically binds to the Cp β2 toxin protein. The monoclonal antibody does not react with other toxins of Clostridium perfringens, thereby ensuring the specificity of the reaction.

[0015] (2) The kit provided by the present invention uses Cp β2 toxin recombinant protein as an antigen and the prepared specific monoclonal antibody as a competitive antibody to prepare a competitive ELISA kit for detecting Cp β2 toxin antibodies in serum. The competitive ELISA kit has higher specificity and sensitivity than the indirect ELISA kit currently used to detect Cp β2 toxin antibodies in serum.

[0016] (3) The kit provided by the present invention uses the monoclonal antibody of Cp β2 recombinant protein as a competitive antibody, which can detect serum antibodies from a variety of animal sources, avoiding the species restriction of sample sources. At present, there are no related products at home and abroad, which fills the technical gap at home and abroad and solves a technical problem that urgently needs to be solved in the production of aquaculture.

[0017] (4) Develop a commercial competitive ELISA test kit for Cp β2 toxin antibodies, which can be used to evaluate the immune effect of the Clostridium difficile triple quadruple vaccine currently used in the sheep industry, and can also be used to conduct serological surveys on sheep that have not been vaccinated with Cp vaccines.

[0018] (5) The technical solution of the present invention is a competitive ELISA technique, which uses mouse monoclonal antibodies as competitive antibodies and is easy to prepare and produce in large quantities. This technology can achieve high-throughput detection and can be operated by relevant professionals according to the instructions, which is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The present invention is a flow chart of the method for preparing the monoclonal antibody that specifically binds to the Cp β2 toxin recombinant protein.

[0020] Figure 2 It is a schematic diagram of the expression of the recombinant protein of Cp β2 toxin of the present invention. The target protein is expressed in a soluble form at about 44 kDa; M: protein marker relative molecular mass standard; 1, 2: induced bacteria supernatant and precipitate; 3, 4: uninduced bacteria supernatant and precipitate; 5, 6: pET-32a empty bacteria supernatant and precipitate.

[0021] Figure 3 This is a schematic diagram of the screening of the induction time of the present invention; M: protein marker relative molecular mass standard; 1-14: supernatant and precipitate at 8h, 10h, 12h, 13h, 14h, 15h, and 16h of induction. As the induction time increases, the protein expression increases and tends to be stable after 12h, so the optimal induction time is 12h.

[0022] Figure 4 This is a schematic diagram of the screening of the final concentration of the IPTG inducer of the present invention; M: protein marker relative molecular mass standard; 1-10: the final concentrations of the IPTG inducer are 1mM, 0.8mM, 0.5mM, 0.25mM, 0.2mM; 11, 12: uninduced bacterial supernatant and precipitate. Different inducer concentrations have no obvious effect on protein expression, so the optimal inducer concentration is 0.2mM.

[0023] Figure 5 It is a schematic diagram of the purification of the recombinant Cp Cp β2 protein of the present invention; M: PM2510 relative molecular mass standard; M: PM2510 relative molecular mass standard; 1: before protein purification; 2: flow-through; 3: 100 mM imidazole; 4: 150 mM imidazole; 5-7: 200 mM imidazole; 8-10: 250 mM imidazole; 11-13: 300 mM imidazole; 14: blank. Low concentration imidazole (100mM, 150mM imidazole) removes impurities, and high concentration imidazole (200mM, 250mM, 300mM imidazole) elutes the target protein.

[0024] Figure 6The figure is a schematic diagram of the purification of the Cp Cp β2 recombinant protein monoclonal antibody of the present invention; M: protein marker relative molecular mass standard; 1: purified Cp β2 recombinant protein monoclonal antibody. The purified ascites showed target bands at 55kDa (heavy chain) and 25kDa (light chain) and the bands were single, indicating that the ascites was successfully prepared and purified. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] Example 1 1. Preparation of Cp β2 recombinant protein According to the gene sequence encoding the Cp β2 toxin protein of Clostridium perfringens obtained from NCBI (GenBank accession number: AYA44279.1), after removing its signal peptide sequence, the full-length 708bp Cp β2 toxin protein encoding gene (CPB) sequence was obtained and sent to Suzhou Hongxun Biotechnology Co., Ltd. for synthesis into pET-32a (+) plasmid. The recombinant plasmid was transformed into competent Escherichia coli BL21 (DE3) pLysS, and after PCR amplification and sequencing identification, the recombinant expression bacteria were obtained. E. coli BL21 (pET32a- cpb 2). E. coli BL21 (pET32a- cpb 2) The strain was cultured at 37°C and 200 rpm until OD 600 When the value was 0.6-0.8, 0.2 mM IPTG was used for induction for 12 h. The induced bacterial solution was centrifuged and precipitated, and the precipitate was washed and resuspended with pH 7.4 PBS, and then the bacteria were ultrasonically broken and centrifuged again. The supernatant was taken to obtain the soluble expressed Cp β2 toxin recombinant protein, which was purified with Ni-NTA Agarose column to obtain the purified Cp β2 toxin recombinant protein; the purity was above 90% after grayscale scanning analysis.

[0028] Nucleotide sequence encoding Clostridium perfringens Cp β2 toxin protein (SEQ ID NO.5): AAGGAAATCGACGCTTAGAAAGGTAATGGAGAATTATCTTAATGCTTT AAAAAACTACGATATTAATACAGTTGTAAATATTTCAGAAGATGAAAGAG TAAAATAATGTTGAACAGTATAGAGAAATGTTAGAGGATTTTAAATCTGAT CCTAACCAACAACTGAAATCTTTTGAAATACTTAATTCACAAAAGAGAGA TAATAAAGAAATATTTAATGTAAAAACTGAATTTTTAAATGGTGCAATTT ATGACATGGAATTTACTGTATCATCTAAAGATGGAAAATTAATAGTATCT AATATGGAAAGAACAAAAATTGAAAATGAGGGGAAATATATTTTAACACC ATCATTTAGAACTCAAGTTTGTACATGGGATGATGAACTAGGACAAGCAA TTGGGGGAGTTTATCCACAAACATATTCTGATAGATTTACATATTATGCA GATAATATATTATTAAACTTCAGACAATATGCAACTTCAGGTTCAAGAGA TTTAAGAGTAGAATATAGTGTTGTAGATCATTGGATGTGGAAAGATGATG TTAAAGCTTCTCAAATGGTATATGGTCAAAATCCTGATTCTGCTAGACAA ATAAGATTATATATAGAAAAAGGACAATCTTTCTATAAATATAGAATAAG AATTAAAAACTTTACACCTGCATCACTTAAAGTTTTTGGTGAAGGATATT GTGCATAG Figure 2 This is a schematic diagram of the expression of Cp β2 recombinant protein; the protein is expressed in a soluble form at around 44 kDa.

[0029] Figure 3 This is a schematic diagram of the screening of induction time; as the induction time increases, the protein expression level increases and tends to be stable after 12 hours, so the optimal induction time is 12 hours.

[0030] Figure 4This is a schematic diagram of the screening of the final concentration of IPTG inducer; different inducer concentrations had no significant effect on protein expression, so the optimal inducer concentration was selected as 0.2mM.

[0031] Figure 5 This is a schematic diagram of the purification of recombinant Cp β2 protein; low concentration imidazole (100mM, 150mM imidazole) removes impurities, and high concentration imidazole (200mM, 250mM, 300mM imidazole) elutes the target protein.

[0032] Figure 6 This is a schematic diagram of the purification of Cp β2 recombinant protein monoclonal antibody; after purification, the ascites fluid showed target bands at 55kDa (heavy chain) and 25kDa (light chain) and the bands were single, indicating that the ascites fluid was successfully prepared and purified.

[0033] 2. Screening of hybridoma cell lines that secrete monoclonal antibodies that specifically bind to Cp β2 recombinant protein Five BALB / c female mice aged 6 to 8 weeks were selected, and the Cp β2 toxin recombinant protein was mixed with Freund's complete adjuvant in a 1:1 ratio for the first mouse immunization; the subsequent immunization was performed every 2 weeks, and the adjuvant used was Freund's incomplete adjuvant, and the volume ratio of Cp β2 recombinant protein to Freund's incomplete adjuvant was 1:1; a total of 4 immunizations. The antigen dose was 100 μg / mouse / time. The serum of the mice was collected 2 weeks after the fourth immunization. The titer of Cp β2 protein antibody in the serum of immunized mice was detected by our homemade indirect ELISA method. P / N ≥ 2.0 was judged as positive, and the titer reached 1:50000 or above. The spleen cells of the mice that qualified for immunization were taken and fused with SP2 / 0 myeloma cells at a fusion ratio of 5:1. The fused cells were half-changed and fully changed on the 7th and 10th days after fusion, respectively. On the third day after the full change of the medium, the hybridoma cells that can secrete anti-Cp β2 toxin protein antibodies were screened by the indirect ELISA method. Replace all the medium in the positive wells obtained in the first screening and perform the second screening. Expand the culture of the wells that are still positive in the second screening and freeze them. Perform subcloning 3 to 5 times continuously until the positive rate is 100%, and freeze the positive cell lines in a liquid nitrogen tank.

[0034] BALB / C mice were sensitized by intraperitoneal injection of 1 mL of autoclaved liquid paraffin. Seven days later, 1.0 × 10 6 The mouse abdominal cavity was observed for changes every day, and when the abdominal cavity was swollen to the point of inconvenience, the ascites was collected. The ascites obtained in the present invention was purified by Thermo Protein G purification column, and then the purity was observed by SDS-PAGE, and the concentration was determined, and then stored at -80°C for later use.

[0035] The hybridoma cell line CPB2-2A4 secreting a monoclonal antibody specifically binding to the Cp β2 recombinant protein of the present invention was deposited in the China Center for Type Culture Collection on October 16, 2024, with the address being Wuhan University, Wuhan, China; the deposit number is CCTCC NO: C2024312, and the scientific description is hybridoma cell line CPB2-2A4 Hybridoma cell line CPB2-2A4; the culture was received and registered by the China Center for Type Culture Collection on October 16, 2024. The viability of the culture was tested by the China Center for Type Culture Collection on October 22, 2024, and the result was viable.

[0036] 3. Preparation method of monoclonal antibody specifically binding to Cp β2 toxin recombinant protein The hybridoma cell line that secretes the monoclonal antibody that specifically binds to the Cp β2 toxin recombinant protein secretes the monoclonal antibody that specifically binds to the Cp β2 toxin recombinant protein. The monoclonal antibody that specifically binds to the Cp β2 toxin recombinant protein specifically binds to the Cp β2 toxin protein and competes with the Cp positive serum to bind to the Cp β2 toxin protein.

[0037] like Figure 1 As shown, the method for preparing a monoclonal antibody that specifically binds to a recombinant protein of Cp β2 toxin comprises: S101, inoculating hybridoma cell line CCTCC NO: C2024312; S102, ascites of BALB / c female mice was obtained, centrifuged, and the middle layer of pale yellow ascites was collected; S103, purifying the ascites to obtain a monoclonal antibody that specifically binds to the Cp β2 toxin recombinant protein.

[0038] Example 2 Establishment of Cp β2 toxin antibody competition ELISA detection method Through the chessboard titration test, the optimal coating concentration of Cp β2 toxin recombinant protein was determined to be 1.5μg / mL, 100μL / well; serum was original, 50μL / well; monoclonal antibody (initial concentration 2mg / ml) was diluted 1:8000, 50μL / well, incubation time 60min; HRP-labeled goat anti-mouse antibody was diluted 1:20000, 100μL / well, incubation time 60min. The substrate solution TMB was 100μL / well, incubation time 15min; stop solution was 50μL / well, and OD450 value was detected immediately after addition.

[0039] The antigen coating procedure is as follows: melt the antigen frozen at -75℃, dilute it to 1.5μg / mL with pH 7.2 phosphate buffer, add it to the ELISA plate, 100μL / well, and transfer it to 4℃ overnight. The next day, discard the antigen solution, wash it, block it with 1% BSA, 100μL / well, incubate it at 37℃ for 90min, and then wash it again. Then add protein stabilizer, 100μL / well, incubate it at room temperature for 10min, spin dry, and blow it in the clean bench for 1h. Finally, put it in the packaging bag and evacuate it.

[0040] The preparation method of the standard positive serum in the kit of the present invention is as follows: select sheep of about 6 months old, mix Cp β2 toxin recombinant protein with Freund's complete adjuvant in a ratio of 1:1 for the first immunization; after an interval of 2 weeks, mix Cp β2 toxin recombinant protein with Freund's incomplete adjuvant in a ratio of 1:1 for the second immunization; after another interval of 2 weeks, mix Cp β2 toxin recombinant protein with Freund's incomplete adjuvant in a ratio of 1:1 for the third immunization; the immunization antigen dosage is 500 μg / sheep / time. Negative serum is collected from healthy sheep of 5-6 months old that have no history of injection of sheep rapid disease, sudden anthrax, lamb dysentery, and enterotoxemia triple quadruple vaccine, and have negative serological tests for brucellosis and tuberculosis, and have negative antibodies to Clostridium perfringens alpha toxin, β1 toxin, β2 toxin, ε toxin, and Clostridium putrefaciens alpha toxin.

[0041] The HRP-labeled goat anti-mouse antibody in the detection kit of the present invention was purchased from Abcam; the monoclonal antibody diluent, enzyme-labeled antibody diluent, TMB colorimetric solution, stop solution, and 50-fold concentrated washing solution were all purchased from Lanzhou Veterinary Research Biological Co., Ltd., Chinese Academy of Agricultural Sciences.

[0042] Example 3 Cp β2 competitive ELISA antibody detection kit Establishment of the test kit: After all reagents are ready, a competitive ELISA kit for detecting Cp β2 toxin antibodies in serum is assembled. The test kit includes: 2 ELISA plates coated with C.pβ2 recombinant antigen (96 wells / block); 1 bottle of 25-fold PBST concentrate (60 mL / bottle); 1 tube of standard positive serum (0.6 mL / tube); 1 tube of standard negative serum (0.6 mL / tube); 1 bottle of monoclonal antibody working solution (12 mL / bottle); 1 bottle of HRP-labeled goat anti-mouse secondary antibody working solution (25 mL / bottle); 1 bottle of TMB substrate colorimetric solution (25 mL / bottle); 1 bottle of stop solution (15 mL / bottle); 2 sheets of sealing film; 1 instruction manual. The kit is stored at 4°C and has a shelf life of 6 months. The kit can detect 180 sera.

[0043] How to use the kit: 1. Take the kit out of the 4℃ refrigerator and equilibrate to room temperature. Dilute the 25x washing solution to 1x working concentration with double distilled water.

[0044] 2. Take out the ELISA plate, determine the required number of ELISA strips according to the number of serum to be tested, and put the remaining strips into the packaging bag and store at 4℃. It is best to use up the unpacked ELISA plate within 10 days.

[0045] 3. Sample addition: 2 wells of standard positive serum and standard negative serum, 50 μL / well; 1 well of the serum sample to be tested, 50 μL / well.

[0046] 4. Add monoclonal antibody working solution, 50 μL / well.

[0047] 5. Incubation: Seal the ELISA plate with a sealing film and incubate it in a 37°C incubator for 60 minutes.

[0048] 6. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard. Repeat this 4 times and pat dry.

[0049] 7. Add HRP-labeled goat anti-mouse secondary antibody working solution, 100 μL / well.

[0050] 8. Incubation: Same as step 5.

[0051] 9. Washing: Same as step 6.

[0052] 10. Color development: Add TMB color development solution, 100 μL / well, and develop color at 37°C in the dark for 15 min.

[0053] 11. Stop: Add stop solution, 50 μL / well.

[0054] 12. Determination: Immediately measure the absorbance (OD) of each well at 450 nm using an ELISA reader. 450 value).

[0055] 13. Result determination ① Calculate the OD of the negative and positive control wells and each serum well to be tested on the same ELISA plate 450 The inhibition rate (PI) of the standard positive serum, negative serum and serum to be tested was calculated according to the following formula. PI = (1-sample OD 450 Average value / standard negative serum OD 450 average value) × 100%.

[0056] ②PI of standard positive serum>80%, OD of standard negative serum 450 >1.0, the test is established, otherwise the test result is invalid.

[0057] ③ For the serum sample to be tested, if PI ≥ 35%, it is positive; if PI < 35%, it is negative.

[0058] Test kit sensitivity, specificity, repeatability, compliance rate, and shelf life determination: Compared with the indirect ELISA kit, the invention has higher sensitivity and specificity in detecting Cp toxin antibodies in serum.

[0059] The standard positive serum of Clostridium perfringens was diluted to 6 dilutions of 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128, and the kit of the present invention was used to detect the samples of each dilution gradient to test the lowest antibody titer that the kit can detect. The results showed that the positive serum could still be detected when diluted to 1:64, indicating that the kit has a high sensitivity.

[0060] The kit was used to detect sera positive for alpha toxin antibody of Clostridium perfringens, positive for β1 toxin antibody, positive for ɛ toxin antibody, positive for alpha toxin antibody of Clostridium putrefaciens, positive for Escherichia coli and positive for Salmonella, and the results were all negative, indicating that there was no cross-reaction between the Cp β2 antigen used in the kit and the positive sera of other Clostridial toxins infecting sheep and common bacteria (Escherichia coli and Salmonella), and the kit had good specificity.

[0061] 20 Cp-positive sera and 20 Cp-negative sera were tested three times under the same conditions at different times using the same batch of test kits, and the results were exactly the same; the sera were tested using 3 batches of test kits, and the results were exactly the same; these results indicate that the test kit has good repeatability.

[0062] The kit was used to test 50 positive sera and 50 negative sera identified by the indirect ELISA kit to verify the consistency of the kit with the indirect ELISA kit. The results showed that the consistency of the kit with the indirect ELISA kit was 97% (97 / 100).

[0063] The same 20 positive sera and 20 negative sera were tested every 2 weeks using the kit prepared from the same batch. The results showed that the kit stored for 6 months gave the same test results for the same serum, indicating that the shelf life of the kit is at least 6 months.

[0064] Example 4 Test kit for detecting field serum samples A total of 200 samples of sheep sera collected from 4 farms were tested using the kit of the present invention. The test results are shown in Table 1.

[0065] From the test results, it can be seen that the kit of the present invention and the indirect ELISA kit were used to detect the above 200 field sera respectively, and the compliance rate of the two methods was 92% (66+118 / 200), indicating that the kit involved in the present invention can completely replace the indirect ELISA kit to detect Cp β2 toxin serum antibodies and be promoted and applied in production.

[0066] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A hybridoma cell line secreting a monoclonal antibody that specifically binds to a recombinant protein of Clostridium perfringens β2, characterized in that: The hybridoma cell line CPB2-2A4 was deposited in the China Center for Type Culture Collection on October 16, 2024, with the deposit number CCTCC NO: C2024312.

2. A monoclonal antibody that specifically binds to a recombinant protein of Clostridium perfringens β2 toxin, characterized in that: The heavy chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.

2.

3. The monoclonal antibody that specifically binds to the recombinant protein of Clostridium perfringens β2 toxin according to claim 2, characterized in that: The heavy chain nucleotide sequence encoding the monoclonal antibody is shown in SEQ ID NO.3, and the light chain nucleotide sequence encoding the monoclonal antibody is shown in SEQ ID NO.

4.

4. The monoclonal antibody that specifically binds to the recombinant protein of Clostridium perfringens β2 toxin according to claim 2 or 3, characterized in that: The monoclonal antibody is produced by a hybridoma cell line with a deposit number of CCTCC NO: C2024312.

5. Use of the monoclonal antibody according to claim 2 in the preparation of a reagent or a kit for detecting antibodies against Clostridium perfringens β2 toxin.

6. A competitive ELISA kit for detecting Clostridium perfringens β2 toxin antibodies in serum, characterized in that: The kit comprises the monoclonal antibody according to claim 2.

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