Monoclonal antibodies, hybridoma cell lines, and applications that specifically bind to recombinant Clostridium perfringens β2 toxin protein

Abstract

This invention belongs to the field of biotechnology and discloses a monoclonal antibody that specifically binds to the recombinant protein of Clostridium perfringens (Cp) β2 toxin, a hybridoma cell line, and its applications. This invention provides a monoclonal antibody that specifically binds to the β2 toxin protein and a hybridoma cell line CPB2-2A4 (accession number CCTCC NO: C2024312) that secretes this monoclonal antibody. The monoclonal antibody reacts only with the Cp β2 toxin protein and does not cross-react with other pathogens infecting cattle and sheep, exhibiting extremely high specificity. The kit utilizes competitive ELISA technology, using a mouse monoclonal antibody that specifically binds to the Cp β2 toxin recombinant protein as a competing antibody, enabling the detection of Cp β2 toxin antibodies in serum from various animal sources, avoiding species limitations of sample sources. Furthermore, the kit exhibits good reproducibility. The kit has relatively relaxed technical requirements during operation and can be widely used in production for applications such as Cp epidemiological surveys and vaccine immunization level monitoring.

Description

Technical Field

[0001] This invention relates to a monoclonal antibody that specifically binds to the recombinant protein of Clostridium perfringens (Cp) β2 toxin, a hybridoma cell line, and their applications, belonging to the field of biotechnology. Background Technology

[0002] Clostridium perfringens ( Clostridium perfringens C. pylori (C. pylori) is an obligate anaerobic Gram-positive bacterium that is part of the normal gut flora of humans and animals. When the host's internal environment changes, it can cause various diseases in humans and animals, such as gas gangrene and human-induced food poisoning, piglet diarrhea, calf enterotoxemia, lamb dysentery, and necrotic enteritis in poultry. The main pathogenic factors of this bacterium are more than 20 exotoxins and hydrolytic enzymes secreted by it, including α, β, ε, and ι. Mature toxins are absorbed by the intestinal mucosa and diffuse through the bloodstream to various tissues and organs, such as the kidneys, lungs, liver, and brain. It can cause enterotoxemia in animals (goats, sheep, and less commonly cattle), leading to rapid death and significant economic losses. Therefore, the prevention and control of this disease is of paramount importance.

[0003] Clostridium perfringens β2 toxin is encoded by the cpb2 gene, and all seven toxin types (AG type) of Clostridium perfringens produce β2 toxin. In 1997, Gibert first identified Clostridium perfringens β2 toxin, which has an isoelectric point (pI) between 5.4 and 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 shows no significant 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 includes a 30-amino acid signal peptide, which is cleaved during β2 toxin transcription. Therefore, the mature β2 toxin protein is a peptide chain of 235 amino acids, with a molecular weight of 27.67 kDa and an isoelectric point of 5.01. β2 toxins possess perforating toxin activity, capable of damaging the cell membranes of various host cells. They can also affect nerve tissue function and disrupt normal nerve conduction by influencing the distribution of calcium ions within nerve cell membranes. They play a significant role in gastrointestinal diseases in animals; for example, beta2-producing *Clostridium perfringens* strains can cause necrotic enteritis in piglets and enterocolitis in horses. Therefore, the role of β2 toxin proteins is crucial in the development of next-generation *Clostridium perfringens* genetically engineered vaccines. Vaccination is the most effective means of preventing *Clostridium perfringens* infection. Serological testing methods are essential for evaluating antibody levels against various *Clostridium perfringens* toxin proteins in the serum of vaccinated animals. This is vital for verifying the quality of *Clostridium perfringens* vaccines and developing high-quality vaccines. However, currently, there are no commercially available ELISA kits for detecting *Clostridium perfringens* β2 toxin antibodies in China. In previous studies, our laboratory established an indirect ELISA method for detecting Cp β2 toxin antibodies in sheep serum. This method is highly specific and sensitive, and can be used to determine the level of Cp β2 toxin antibodies in serum. However, indirect ELISA has a relatively high false positive rate and only detects antibody levels in the serum of specific animals. Therefore, it is necessary to establish a method that can detect Cp β2 toxin antibody levels in the serum of multiple animals.

[0004] Monoclonal antibody technology involves fusing antibody-producing single B lymphocytes with bone marrow tumor cells to obtain hybrid cells that can both produce antibodies and proliferate indefinitely, thereby producing highly specific monoclonal antibodies. 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 requiring relatively low sample purity and allowing detection even in complex sample matrices. Competitive ELISA is simple to operate, low in cost, and suitable for detecting large quantities of serum samples from various animals, making it suitable for large-scale screening. The preparation of a monoclonal antibody against Clostridium perfringens β2 toxin protein, and the development of a competitive ELISA kit for detecting Clostridium perfringens β2 toxin antibodies using this monoclonal antibody, will have significant application value for the prevention and control of Clostridium perfringens infection. Summary of the Invention

[0005] The purpose of this invention is to provide a hybridoma cell line CPB2-2A4 that can secrete a monoclonal antibody that specifically binds to the 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 objective of this invention is to provide a competitive ELISA kit for detecting Clostridium perfringens β2 toxin antibodies in serum.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] This invention provides a hybridoma cell line CPB2-2A4 that specifically binds to a monoclonal antibody against Clostridium perfringens β2 recombinant protein. It was deposited on October 16, 2024, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China; accession number CCTCC NO: C2024312, and classification name: Hybridoma cell line CPB2-2A4.

[0010] This invention provides a monoclonal antibody that specifically binds to the recombinant protein of Clostridium 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.

[0011] Heavy chain variable region amino acid sequence (SEQ ID NO.1):

[0012] QIQLVQSGPELKKPGETVKFSCKASGYTFTNYGMNWMKQAPGKGLKWMGWINTYTGEATYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARDYYGSTYGDFEYWGQGTTLTVSS

[0013] Light chain variable region amino acid sequence (SEQ ID NO.2):

[0014] DIVLTQSPASLAVSLGQRATISCRASQSVSASTFSSIHWYQQKPGQPPKLLIKYASDLKSGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWQIPYTFGGGTKLAIKRA

[0015] 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.

[0016] Heavy chain variable region nucleotide sequence (SEQ ID NO.3):

[0017] CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGTTCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGAACTGGATGAAACAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGGCAACATATGC TGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGAGATTACTACGGTAGTACCTACGGAGACTTTGAATACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGC

[0018] Light chain variable region nucleotide sequence (SEQ ID NO.4):

[0019] GACATTTGTGCTGACTCAGTCTCCTGCTTCCTTAGCTGTTTCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGCCAAAGTGTCAGTGCATCTACCTTTAGTTCTATACACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCAAGTATGCATCCG ACCTCAAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATACTGCAACATATTACTGTCAGCACAGTTGGCAGATTCCCTATACGTTCGGAGGGGGGACCAAGCTTGGCGATAAAACGGGCT

[0020] A monoclonal antibody specifically binding to the recombinant Cp β2 toxin protein was produced from a hybridoma cell line with accession number CCTCC NO:C2024312. The monoclonal antibody specifically binds to the recombinant Cp β2 toxin protein and competitively binds to it in Cp-positive serum. The preparation method of the monoclonal antibody specifically binding to the recombinant Cp β2 toxin protein was as follows: The hybridoma cell line CCTCC NO:C2024312 was intraperitoneally injected into female BALB / c mice. Ascites fluid was obtained from the female BALB / c mice, centrifuged, and the pale yellow intermediate layer was collected. After purification, the monoclonal antibody specifically binding to the recombinant Cp β2 toxin protein was obtained.

[0021] This invention provides the application of monoclonal antibodies in the preparation of reagents or kits for detecting Clostridium perfringens Cp β2 toxin antibodies in serum.

[0022] This 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 using recombinant Cp β2 toxin protein as the antigen and a monoclonal antibody that specifically binds to the recombinant Cp β2 toxin protein as the competitive antibody.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention provides a monoclonal antibody that specifically binds to the Cp β2 toxin protein and a hybridoma cell line, CPB2-2A4 (CCTCC NO: C2024312), that secretes this monoclonal antibody. The monoclonal antibody secreted by this cell line reacts only with the Cp β2 toxin protein and does not cross-react with other pathogens infecting cattle and sheep, exhibiting extremely high specificity. The kit utilizes competitive ELISA technology, using a mouse monoclonal antibody that specifically binds to the recombinant Cp β2 toxin protein as a competing antibody, enabling the detection of serum antibodies from multiple animal sources and avoiding species limitations of sample sources. Furthermore, the kit exhibits good reproducibility. The kit has relatively relaxed technical requirements during operation and can be widely used in production for applications such as Cp epidemiological surveys and vaccine immunization level monitoring.

[0025] (1) In this invention, soluble recombinant Cp β2 toxin protein was used as an antigen to immunize mice. Mouse spleen cells were then fused with myeloma SP2 / 0 cells, and a hybridoma cell line CPB2A4 secreting a specific monoclonal antibody was screened. Its preservation number is CCTCC NO:C2024312. In this invention, the CCTCC NO:C2024312 cell line was inoculated into the peritoneal cavity of mice, and the ascites fluid was collected to purify the antibody, obtaining a purified monoclonal antibody that specifically binds to the Cp β2 toxin protein. This monoclonal antibody does not react with other toxins of Clostridium perfringens, ensuring the specificity of the reaction.

[0026] (2) The kit provided by the present invention uses recombinant Cp β2 toxin protein as antigen and prepared specific monoclonal antibody as competitive antibody to prepare a competitive ELISA kit for detecting Cp β2 toxin antibody in serum. It has higher specificity and sensitivity than the indirect ELISA kit currently used to detect Cp β2 toxin antibody in serum.

[0027] (3) The kit provided by this invention uses a monoclonal antibody against Cpβ2 recombinant protein as a competitive antibody, which can detect serum antibodies from a variety of animal sources, avoiding species limitations of sample sources. Currently, there are no related products at home and abroad, filling a technological gap and solving a technical problem that urgently needs to be solved in aquaculture production.

[0028] (4) Develop a commercial Cp β2 toxin antibody competitive ELISA detection kit to evaluate the immunization effect of the Clostridium trivalent and quadrivalent vaccine currently used in sheep farming, and to conduct serological surveys on sheep that have not been immunized with Cp vaccine.

[0029] (5) The technical solution of the present invention is a competitive ELISA technology, which uses murine 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, making it easy to operate. Attached Figure Description

[0030] Figure 1 This is a flowchart of the preparation method of the monoclonal antibody that specifically binds to the recombinant Cpβ2 toxin protein according to the present invention.

[0031] Figure 2 This is a schematic diagram of the expression of the recombinant Cp β2 toxin protein of the present invention. The target protein is expressed in soluble form at around 44 kDa; M: relative molecular mass standard of protein marker; 1, 2: supernatant and precipitate of induced bacteria; 3, 4: supernatant and precipitate of uninduced bacteria; 5, 6: supernatant and precipitate of pET-32a empty vector bacteria.

[0032] Figure 3 This is a schematic diagram of the induction time selection for this invention; M: relative molecular mass standard of protein marker; 1~14: supernatant and precipitate after 8h, 10h, 12h, 13h, 14h, 15h, and 16h of induction. As the induction time increases, the protein expression level increases, and it tends to stabilize after 12h; therefore, the optimal induction time is 12h.

[0033] Figure 4 This is a schematic diagram illustrating the screening of the final concentration of the IPTG inducer in this invention; M: relative molecular mass standard of protein marker; 1~10: final concentrations of IPTG inducer are 1 mM, 0.8 mM, 0.5 mM, 0.25 mM, and 0.2 mM, respectively; 11, 12: supernatant and precipitate of uninduced bacteria. Different inducer concentrations have no significant effect on protein expression levels, therefore the optimal inducer concentration is selected as 0.2 mM.

[0034] Figure 5 This is a schematic diagram of the purification process of recombinant CpCpβ2 protein according to the present invention; M: PM2510 relative molecular mass standard; 1: Protein before purification; 2: Flow-through buffer; 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 concentrations of imidazole (100 mM, 150 mM imidazole) are used to remove impurities, while high concentrations of imidazole (200 mM, 250 mM, 300 mM imidazole) are used to elute the target protein.

[0035] Figure 6This is a schematic diagram illustrating the purification of the Cp β2 recombinant protein monoclonal antibody of this invention; M: relative molecular mass standard of the protein marker; 1: purified Cp β2 recombinant protein monoclonal antibody. The purified ascites fluid showed the target bands at 55 kDa (heavy chain) and 25 kDa (light chain), and the bands were single, indicating successful preparation and purification of the ascites fluid. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0037] Example 1

[0038] 1. Preparation of Cpβ2 recombinant protein

[0039] Based on the gene sequence encoding the Clostridium perfringens Cp β2 toxin protein obtained from NCBI (GenBank accession number: AYA44279.1), after removing its signal peptide sequence, the full-length 708 bp Cp β2 toxin protein encoding gene (CPB) sequence was obtained and sent to Suzhou Hongxun Biotechnology Co., Ltd. to synthesize it into the pET-32a (+) plasmid. The recombinant plasmid was transformed into competent Escherichia coli BL21(DE3)pLysS, and after PCR amplification and sequencing confirmation, the recombinant expression strain was obtained. E. coli BL21 (pET32a- cpb 2). E. coli BL21 (pET32a- cpb 2) The strain was cultured at 37℃ and 200 r / min until OD. 600 When the pH value was 0.6–0.8, the cells were induced with 0.2 mM IPTG for 12 h. After induction, the bacterial culture was centrifuged to precipitate, washed with pH 7.4 PBS, resuspended, and then the cells were sonicated to disrupt the bacterial cells. After centrifugation, the supernatant was collected to obtain soluble recombinant Cp β2 toxin protein. The purified Cp β2 toxin recombinant protein was obtained by purification using a Ni-NTA agarose column. The purity was above 90% according to grayscale analysis.

[0040] The nucleotide sequence encoding the Clostridium perfringens Cp β2 toxin protein (SEQ ID NO.5):

[0041] AAGGAAATCGACGCTTAGAAAGGTAATGGAGAATTATCTTAATGCTTT

[0042] AAAAAACTACGATTAATACAGTTGTAAATATTTCAGAAGATGAAAGAG

[0043] TAAATAATGTTGAACAGTAGAAATGTTAGAGGATTTTAAATCTGAT

[0044] CCTAACCAACAACTGAAATCTTGAAATACTTAATTCAAAAGAGAGA

[0045] TAATAAAGAAATATTTAATGTAAAAACTGAATTTTTAAATGGTGCAATTT

[0046] ATGACATGGAATTTACTGTATCATCTAAAGATGGAAAATTAATAGTATCT

[0047] AATATGGAAAGAACAAAATTGAAAATGAGGGAAATATTTTAACACC

[0048] ATCATTTAGAACTCAAGTTTGTACATGGGATGATGAACTAGGACAAGCAA

[0049] TTGGGGGAGTTTCCACAAACATATTCTGATAGATTTACATATTATGCA

[0050] GATAATATTATTAAACTTCAGACAATATGCAACTTCAGGTTCAAGAGA

[0051] TTTAAGTAGAATAGTGTTGTAGATCATTGGATGTGGAAAGATGATG

[0052] TTAAAGCTTCTCAAATGGTATATGGTCAAAATCCTGATTCTGCTAGACAA

[0053] ATAAGATTATATAGAAAAAGGACAATCTTTCTATAAATATAGAATAAG

[0054] AATTAAAAACTTTACACCTGCATCACTTAAAGTTTTTGGTGAAGGATATT

[0055] GTGCATAG

[0056] Figure 2 This is a schematic diagram of the expression of Cpβ2 recombinant protein; the protein is expressed in soluble form at around 44 kDa.

[0057] Figure 3 This is a schematic diagram of the induction time selection; as the induction time increases, the protein expression level increases and tends to stabilize after 12 h, so the optimal induction time is 12 h.

[0058] Figure 4 This is a schematic diagram of the screening of the final concentration of IPTG inducer; different inducer concentrations have no significant effect on protein expression levels, so the optimal inducer concentration is selected as 0.2 mM.

[0059] Figure 5 This is a schematic diagram of the purification of recombinant Cp β2 protein; low concentrations of imidazole (100mM, 150mM imidazole) remove impurities, while high concentrations of imidazole (200mM, 250mM, 300mM imidazole) elute the target protein.

[0060] Figure 6 This is a schematic diagram of the purification of Cpβ2 recombinant protein monoclonal antibody; the purified ascites fluid showed the 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.

[0061] 2. Screening of hybridoma cell lines that secrete monoclonal antibodies that specifically bind to Cpβ2 recombinant protein

[0062] Five 6-8 week old female BALB / c mice were selected. A 1:1 mixture of recombinant Cp β2 toxin protein and Freund's complete adjuvant was used for the first immunization. Subsequent immunizations were performed every two weeks using Freund's incomplete adjuvant, with a 1:1 volume ratio of recombinant Cp β2 protein to Freund's incomplete adjuvant. A total of four immunizations were administered. The antigen dose was 100 μg / mouse / immunization. Serum was collected two weeks after the fourth immunization. The titer of Cp β2 protein antibodies in the immunized mouse serum was detected using a self-developed indirect ELISA method. A P / N ratio ≥ 2.0 was considered positive, and a titer of 1:50000 or higher was considered immunization successful. Spleen cells from immunized mice were fused with SP2 / 0 myeloma cells at a ratio of 5:1. The fused cells underwent partial and complete medium replacements on days 7 and 10 post-fusion, respectively. On day three after the complete medium replacement, hybridoma cells secreting anti-Cp β2 toxin protein antibodies were screened using an indirect ELISA method. For the positive wells identified in the first screening, completely replace the medium and perform a second screening. Expand the culture of the wells that are still positive in the second screening and freeze them. At the same time, perform subcloning 3-5 times continuously until the positive rate is 100%, and freeze the positive cell lines in liquid nitrogen.

[0063] BALB / c mice were sensitized by intraperitoneal injection of 1 mL of autoclaved liquid paraffin, followed by intraperitoneal injection of 1.0 × 10⁻⁶ mol / L paraffin 7 days later. 6 A number of well-growing positive hybridoma cells were collected. The peritoneal cavity of mice was observed daily, and ascites fluid was collected when the mice became distended to the point of difficulty in movement. The ascites fluid obtained in this invention was purified using a Thermo Protein G purification column, and its purity was observed by SDS-PAGE. The concentration was also determined, and the fluid was stored at -80°C for later use.

[0064] The hybridoma cell line CPB2-2A4, which secretes a monoclonal antibody specifically binding to the Cpβ2 recombinant protein, was deposited on October 16, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China; accession number CCTCC NO: C2024312, scientifically described as hybridoma cell line CPB2-2A4. This culture was received by CTCC on October 16, 2024, and registered. The viability of this culture was tested by CTCC on October 22, 2024, and the result was positive.

[0065] 3. Preparation method of monoclonal antibody specifically binding to recombinant Cpβ2 toxin protein

[0066] Hybridoma cell lines secrete monoclonal antibodies that specifically bind to recombinant Cp β2 toxin protein. These monoclonal antibodies specifically bind to the Cp β2 toxin protein and competitively bind to it in Cp-positive serum.

[0067] like Figure 1 As shown, the method for preparing monoclonal antibodies that specifically bind to recombinant Cpβ2 toxin protein includes:

[0068] S101, the hybridoma cell line CCTCC NO:C2024312 was inoculated;

[0069] S102, obtain ascites fluid from female BALB / c mice, centrifuge, and collect the middle layer of pale yellow ascites fluid;

[0070] S103, after purifying ascites fluid, yields a monoclonal antibody that specifically binds to the recombinant Cp β2 toxin protein.

[0071] Example 2: Establishment of a competitive ELISA method for detecting Cp β2 toxin antibodies.

[0072] The optimal coating concentration of Cp β2 toxin recombinant protein was determined to be 1.5 μg / mL, 100 μL / well using a checkerboard titration assay; serum was diluted undiluted, 50 μL / well; monoclonal antibody (initial concentration 2 mg / mL) was diluted 1:8000, 50 μL / well, with an incubation time of 60 min; HRP-labeled goat anti-mouse antibody was diluted 1:20000, 100 μL / well, with an incubation time of 60 min. The substrate solution TMB was 100 μL / well, with an incubation time of 15 min; the stop solution was 50 μL / well, and the OD450 value was measured immediately after addition.

[0073] The antigen coating procedure was as follows: Thaw the antigen stored at -75℃, dilute it to 1.5 μg / mL with pH 7.2 phosphate buffer, add 100 μL / well to the microplate, and incubate overnight at 4℃. The next day, discard the antigen solution, wash, and block with 1% BSA (100 μL / well), incubate at 37℃ for 90 min, then wash again. Next, add protein stabilizer (100 μL / well), incubate at room temperature for 10 min, spin dry, and blow dry in a clean bench for 1 h. Finally, place in a packaging bag and vacuum seal.

[0074] The preparation method of the standard positive serum in the kit of this invention is as follows: Select sheep around 6 months old, mix Cp β2 toxin recombinant protein with Freund's complete adjuvant at a 1:1 ratio for the first immunization; after an interval of 2 weeks, mix Cp β2 toxin recombinant protein with Freund's incomplete adjuvant at a 1:1 ratio for the second immunization; after another interval of 2 weeks, mix Cp β2 toxin recombinant protein with Freund's incomplete adjuvant at a 1:1 ratio for the third immunization; the dose of the immunogen is 500 μg / sheep / time for each immunization. Negative serum is collected from healthy 5-6 month old sheep that have not had a history of vaccination against sheep enterotoxemia, sudden anthrax, lamb dysentery, or enterotoxemia, and whose serological tests for brucellosis and tuberculosis are negative, and whose antibody tests for Clostridium perfringens α toxin, β1 toxin, β2 toxin, ε toxin, and Clostridium putrefactivee α toxin are negative.

[0075] The HRP-labeled goat anti-mouse antibody in the detection kit of this invention was purchased from Abcam; the monoclonal antibody diluent, enzyme-labeled antibody diluent, TMB chromogenic solution, stop solution, and 50-fold concentrated washing solution were all purchased from Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences.

[0076] Example 3: Cpβ2 competitive ELISA antibody detection kit

[0077] Setup of the Detection Kit: After all reagents are prepared, a competitive ELISA kit for detecting Cpβ2 toxin antibodies in serum is assembled. The kit includes: two ELISA plates coated with C.pβ2 recombinant antigen (96 wells / plate); one bottle of 25x PBST concentrate (60 mL / bottle); one tube of standard positive serum (0.6 mL / tube); one tube of standard negative serum (0.6 mL / tube); one bottle of monoclonal antibody working solution (12 mL / bottle); one bottle of HRP-labeled goat anti-mouse secondary antibody working solution (25 mL / bottle); one bottle of TMB substrate chromogenic solution (25 mL / bottle); one bottle of stop solution (15 mL / bottle); two sealing films; and one instruction manual. The kit should be stored at 4°C for 6 months. The kit can test 180 serum samples.

[0078] Instructions for using the kit:

[0079] 1. Remove the kit from the 4°C freezer and allow it to equilibrate to room temperature. Dilute the wash buffer (25x dilution) with double-distilled water to a working concentration of 1.

[0080] 2. Remove the ELISA plate and determine the required number of strips based on the amount of serum to be tested. Place any remaining strips in the packaging bag and store at 4°C. Opened ELISA plates should ideally be used within 10 days.

[0081] 3. Sample addition: Add 50 μL of standard positive serum and standard negative serum to 2 wells each; add 50 μL of the serum sample to be tested to 1 well.

[0082] 4. Add monoclonal antibody working solution, 50 μL / well.

[0083] 5. Incubation: Seal the microplate with sealing film and incubate at 37°C for 60 minutes.

[0084] 6. Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let stand for 30 seconds, then discard. Repeat this process 4 times, then pat dry.

[0085] 7. Add HRP-labeled goat anti-mouse secondary antibody working solution, 100 μL / well.

[0086] 8. Incubation: The procedure is the same as step 5.

[0087] 9. Washing: Same as step 6.

[0088] 10. Color development: Add 100 μL of TMB color development solution to each well and develop at 37°C in the dark for 15 min.

[0089] 11. Termination: Add stop solution, 50 μL / well.

[0090] 12. Measurement: Immediately measure the absorbance (OD) of each well using a microplate reader at a wavelength of 450 nm.450 value).

[0091] 13. Result Determination

[0092] ① Calculate the OD values ​​of the negative and positive control wells and each serum sample well on the same ELISA plate. 450 The inhibition rate (PI) of standard positive serum, negative serum, and test serum is calculated using the following formula: PI = (1 - sample OD) / (sample OD). 450 Mean / Standard Negative Serum OD 450 (Average) × 100%.

[0093] ② The PI of standard positive serum is >80%, and the OD of standard negative serum is >80%. 450 If the result is greater than 1.0, the experiment is successful; otherwise, the result of the experiment is invalid.

[0094] ③ For the serum sample to be tested, a PI ≥ 35% is considered positive; a PI < 35% is considered negative.

[0095] The kit's sensitivity, specificity, repeatability, concordance rate, and shelf life were determined.

[0096] Compared with indirect ELISA kits, the present invention has higher sensitivity and specificity in detecting Cp toxin antibodies in serum.

[0097] Clostridium perfringens standard positive serum was diluted to six levels: 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128. Samples at each dilution were tested using the kit described in this invention to verify the lowest detectable antibody titer. The results showed that the positive serum could still be detected at a dilution of 1:64, indicating that the kit has high sensitivity.

[0098] The kit was used to detect positive sera for Clostridium perfringens α-toxin antibody, β1-toxin antibody, α-toxin antibody, Clostridium putrefactivee α-toxin antibody, Escherichia coli, and Salmonella. All results were negative, indicating that there is no cross-reactivity between the Cp β2 antigen used in the kit and positive sera for other Clostridium toxins and common bacteria (Escherichia coli and Salmonella) in infected sheep, and the kit has good specificity.

[0099] Twenty Cp-positive serum samples and twenty Cp-negative serum samples were tested three times using the same batch of kits at different times and under the same conditions, and the results were completely identical. The above serum samples were tested using three batches of kits, and the results were completely identical. These results indicate that the kits have good repeatability.

[0100] Fifty positive and fifty negative serum samples identified by the indirect ELISA kit were tested using the kit to verify the concordance rate between the kit and the indirect ELISA kit. The results showed that the concordance rate between the kit and the indirect ELISA kit was 97% (97 / 100).

[0101] Twenty positive and twenty negative serum samples were tested using kits prepared in the same batch, with a two-week interval between each test. The results showed that kits stored for six months produced identical results for the same serum sample, indicating that the kits have a shelf life of at least six months.

[0102] Example 4: Reagent kit for detecting field serum samples

[0103] The reagent kit of this invention was used to test 200 sheep serum samples collected from four farms. The test results are shown in Table 1.

[0104]

[0105] The test results show that when the above 200 field serum samples were tested using the kit of this invention and the indirect ELISA kit, the concordance rate of the two methods was 92% (66+118 / 200). This indicates that the kit of this invention can completely replace the indirect ELISA kit for detecting Cp β2 toxin serum antibodies and can be widely used in production.

[0106] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

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

2. A monoclonal antibody that specifically binds to the recombinant protein of Clostridium perfringens β2 toxin, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region 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 monoclonal antibody was produced by a hybridoma cell line with accession number CCTCC NO:C2024312.

4. The use of the monoclonal antibody as described in claim 2 in the preparation of reagents or kits for detecting Clostridium perfringens β2 toxin antibodies.

5. A competitive ELISA kit for detecting Clostridium perfringens β2 toxin antibodies in serum, characterized in that: The kit includes the monoclonal antibody as described in claim 2.

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