Clostridium perfringens ETX antibody blocking ELISA detection method

Abstract

The invention belongs to the technical field of biology, and particularly relates to a hybridoma cell strain capable of secreting a clostridium perfringens ETX monoclonal antibody with neutralizing activity and application of the hybridoma cell strain. On the basis of natural toxin, an indirect ELISA (enzyme-linked immunosorbent assay) detection method is established by utilizing a non-toxic ETX single amino acid site protein mutant, and a series of hybridoma cell strains secreting an anti-clostridium perfringens ETX monoclonal antibody are screened; the hybridoma cell strain can stably and efficiently secrete a monoclonal antibody with neutralizing activity and a monoclonal antibody without neutralizing resistance respectively, and large-scale batch production can be realized. According to the present invention, the clostridium perfringens ETX monoclonal antibody is firstly utilized to establish the ETX antigen ELISA detection method, and the method has characteristics of simple sample operation, low cost, rapid reaction, strong specificity and the like, can provide the reference for the diagnosis of clostridium perfringens, and provides the basis for the research of the related vaccine potency test substitution method.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a hybridoma cell line secreting a neutralizing active Clostridium perfringens ETX monoclonal antibody and its application. Background Art

[0002] Clostridium perfringens (CP) is a Gram-positive, spore-forming anaerobic bacillus that survives in a strictly anaerobic environment, can produce spores, has strong environmental adaptability, and is widely present in the intestines of humans and animals. It can cause various systemic and gastrointestinal diseases in humans and animals, including human gas gangrene, food poisoning, gastrointestinal diseases, and liver and kidney damage, as well as gangrenous dermatitis in animals, necrotic enteritis (NE) in cattle, sheep, pigs, and poultry, and enterotoxemia in cattle and sheep. Clostridium perfringens can produce more than 20 toxins. According to the differences in the main lethal toxins alpha (CPA), beta (CPB), epsilon (ETX), iota (ITX), Clostridium perfringens enteroloxin (CPE), and Neerlie enleritis toxin B (NetB), Clostridium perfringens is divided into 7 toxin types A - G (ROOD JI, ADAMS V, LACEY J, et al. Expansion of the Clostridium perfringens toxin-based typing scheme[J]. Anaerobe. 2018 Oct;53:5 - 10.). Among them, type A Clostridium perfringens mainly causes animal enterotoxemia, gas gangrene, and necrotic enteritis, etc.; types B, C, D, and E Clostridium perfringens mainly cause various diseases such as lamb dysentery, sheep struck, and enterotoxemic hemorrhage, and type C can also cause human necrotic enteritis; type F can cause human foodborne diseases and lead to watery diarrhea; type G is closely related to chicken necrotic enteritis (XU Jirong, ZHAO Cui, FAN Li, et al. Analysis of drug resistance and molecular genetic evolution characteristics of Clostridium perfringens strains isolated from sheep in Shandong Province[J]. Chinese Journal of Preventive Veterinary Medicine, 2024, 46(6):593 - 600.).

[0003] ETX is produced by Clostridium perfringens type B and type D, and is the third most potent clostridial toxin after botulinum and tetanus neurotoxins. It is also the most virulent toxin among all the exotoxins of Clostridium perfringens. The full-length of this toxin is 296 amino acids and is secreted outside the bacteria in the form of a toxin precursor (HUNTER S E, CLARKE IN, KELLYD C, et al. Cloning and nucleotide sequencing of the Clostridium perfringens epsilon-toxin gene and its expression in Escherichia coli[J]. Infect Immun, 1992, 60(1):102–110.). After the toxin precursor is acted on by host trypsin, chymotrypsin or the protease of Clostridium itself, 11 - 13 amino acids at the N-terminus and 22 - 29 amino acids at the C-terminus are removed, thus being activated into the mature toxin (FREEDMAN J C, MCCLANE B A, UZAL F A. New insights into Clostridium perfringens epsilon toxin activation and action on the brain during enterotoxemia[J]. Anaerobe, 2016, 41:27-31.). This toxin belongs to the pore-forming toxin family and is mainly divided into three regions, namely I, II and III, which play important roles in the process of toxin binding to cell receptors, maintaining the stability of toxin binding to cell receptors, and forming cell membrane pores. Since two peptide chains of this toxin cross the three regions of I, II and III at the same time, the strategy of using only one domain of the toxin as a vaccine candidate antigen (such as the C-terminus of α and β toxins) is difficult to apply to the prevention and control of this toxin (DU Jige, PENG Xiaobing, ZHANG Xiukun, et al. Expression and immunoprotective evaluation of Clostridium perfringens epsilon toxin mutants[J]. Chinese Journal of Veterinary Drug, 2018, 52(6):13-20.). The virulence of ETX is second only to botulinum toxin and tetanus toxin, and can cause severe enterotoxemia in poultry and livestock, especially ruminants, causing serious economic losses to the livestock industry. Therefore, achieving the attenuation or even detoxification of ETX while retaining its immunogenicity, and then preparing appropriate biological agents such as vaccines, is of great significance in terms of economy and biosafety. The soluble recombinant ETX obtained by Li Qing et al. through the prokaryotic system has strong virulence, resulting in biosafety hazards such as toxin leakage or incomplete inactivation during the preparation of the wild-type recombinant ETX subunit vaccine (LI Qing. Construction and application of Clostridium perfringens α and ε toxin mutants[D]. Anhui: Anhui Medical University, 2013.).

[0004] At present, traditional natural toxin inactivated vaccines are usually used for immunoprophylaxis of animals such as cattle, sheep and rabbits, but there are lack of relevant toxin detection and treatment methods. Due to the complex antigen components and low effective content of natural toxin inactivated vaccines, they are prone to cause side reactions in animals, resulting in their inapplicability for the prevention of animals such as dogs and pigs. Existing research has shown that the preparation of monoclonal antibodies can provide a basis for the establishment of the treatment and diagnosis of certain toxins. The key to monoclonal antibody preparation is the immunogen and the coating antigen, but the preparation, purification and inactivation processes of natural toxins are complex and have high biosafety risks. Summary of the Invention

[0005] Based on the selection of natural toxins, the present invention uses a non-toxic ETX single amino acid site (histidine at position 106 mutated to proline) protein mutant (rETX m1 ) to establish an indirect ELISA detection method, and a series of hybridoma cell lines secreting monoclonal antibodies against Clostridium perfringens ETX are screened, thus providing a material basis for the later detection of ETX antigen.

[0006] In the first aspect, the present invention provides a hybridoma cell line secreting monoclonal antibodies against Clostridium perfringens ETX, and the hybridoma cell line has a preservation number of CGMCC NO.46042, and the monoclonal antibody secreted by the cell line has neutralizing activity.

[0007] In the second aspect, the present invention provides a screening method for the hybridoma cell line described in the first aspect, and the screening method includes the following steps:

[0008] S1. Immunize mice with natural ETX, determine the serum titer by ELISA method, and use the spleen cells of the mouse with the highest titer for cell fusion;

[0009] S2. Fuse the mouse spleen cells screened in S1 with myeloma cells;

[0010] S3. Screen positive clones and subclones using recombinant ETX to obtain a hybridoma cell line that can secrete monoclonal antibodies against Clostridium perfringens ETX with neutralizing activity.

[0011] Further, in step S1, the natural ETX refers to the inactivated natural ETX of Clostridium perfringens.

[0012] Further, in step S2, the myeloma cells refer to SP2 / 0 cells.

[0013] Further, in step S2, the fusion method is the PEG-mediated chemical method.

[0014] Further, in step S3, the recombinant ETX is a non-toxic ETX single amino acid site (histidine at position 106 mutated to proline) protein mutant (rETX m1 ).

[0015] Further, in step S3, the positive clone screening refers to screening positive hybridoma cells by an indirect ELISA method using rETX m1 as the coated antigen.

[0016] Further, in step S3, the subclone screening refers to performing a serum neutralization experiment on the cell supernatant of positive hybridoma cells with the prepared natural toxin of Clostridium perfringens, and screening for hybridoma cell lines with neutralizing activity after subcloning.

[0017] In a third aspect, the present invention provides an antibody against Clostridium perfringens ETX, and the antibody is a monoclonal antibody or a polyclonal antibody; the monoclonal antibody is obtained from the culture solution of the hybridoma cell line described in the first aspect, or obtained by inoculating the hybridoma cell line described in the first aspect into the abdominal cavity of a mouse to produce ascites; the polyclonal antibody is the serum obtained by immunizing a mouse with inactivated natural ETX and then collecting blood and centrifuging.

[0018] In a fourth aspect, the present invention provides an antibody blocking ELISA detection method for Clostridium perfringens ETX, and in the method, the coated antigen is selected as the ETX four amino acid mutant rETX m4 , the primary antibody is the polyclonal antibody described in the third aspect, and the secondary antibody is a monoclonal antibody labeled with HRP.

[0019] Further, the ETX four amino acid mutant rETX m4 is obtained by mutating the amino acids at positions 30 (tyrosine mutated to alanine), 106 (histidine mutated to proline), 196 (tyrosine mutated to alanine), and 199 (phenylalanine mutated to glutamate) of ETX, and the obtained ETX mutant (rETX m4 ).

[0020] Further, the monoclonal antibody labeled with HRP is a monoclonal antibody obtained by purifying the ascites of the monoclonal antibody described in the third aspect and then performing HRP conjugation labeling.

[0021] Further, the working dilution of the ETX polyclonal antibody is 1:500, and the working concentration of the monoclonal antibody labeled with HRP conjugate is 1:8000.

[0022] In a fifth aspect, the present invention provides an application of the hybridoma cells described in the first aspect or the antibody described in the third aspect in the preparation of a reagent for detecting Clostridium perfringens ETX.

[0023] In a sixth aspect, the present invention provides a kit for detecting Clostridium perfringens ETX, and the kit contains the antibody described in the third aspect.

[0024] Further, the kit can be one or more of an ELISA detection kit, a colloidal gold detection kit, an immunohistochemistry kit, an immunofluorescence kit, and / or an in situ hybridization staining kit.

[0025] Furthermore, the diagnostic methods of the kit include one or more of a direct method, an indirect method, a double antibody sandwich method, and / or a competitive method.

[0026] The hybridoma cell line secreting monoclonal antibodies against ETX in the present invention has been deposited with the China General Microbiological Culture Collection Center (CGMCC). The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. The classification name is murine hybridoma cell, and the deposit number is CGMCC NO.46042. The deposit date is August 19, 2024.

[0027] The hybridoma cell line secreting monoclonal antibodies against ETX in the present invention has been deposited with the China General Microbiological Culture Collection Center (CGMCC). The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. The classification name is murine hybridoma cell, and the deposit number is CGMCC NO.46043. The deposit date is August 19, 2024.

[0028] Beneficial effects

[0029] 1. The present invention first uses natural ETX as an immunizing antigen, uses a non-toxic single amino acid ETX mutant as a coating antigen, and combines the cell neutralization test of Clostridium perfringens natural toxin and the serum neutralization test of mice to screen monoclonal antibody hybridoma cell lines with neutralizing activity. The implementation of the above methods has the following advantages:

[0030] (1) Using a small amount of natural ETX as an immunizing antigen ensures that the natural conformation of ETX is maintained to the greatest extent.

[0031] (2) Using a non-toxic ETX mutant with a purity of more than 90% as a coating antigen to establish an ELISA detection method, and screening positive clones and subclones, which not only ensures the similarity between the coating antigen and the natural toxin, but also avoids the cumbersome purification steps and biosafety hazards of Clostridium perfringens natural ETX.

[0032] (3) Using the natural toxin of Clostridium perfringens for the cell neutralization test and serum neutralization test of monoclonal antibodies, so as to obtain monoclonal antibody hybridoma cell lines with neutralizing resistance to natural ETX.

[0033] 2. Obtain a hybridoma cell line of Clostridium perfringens ETX monoclonal antibody that secretes neutralizing-resistant antibodies. This hybridoma cell line can stably and efficiently secrete monoclonal antibodies with neutralizing activity and can achieve large-scale batch production.

[0034] 3. For the first time, use a Clostridium perfringens ETX monoclonal antibody with neutralizing resistance to bind to a four-amino acid mutant of ETX (rETX m4 ) to establish an antibody-blocking ELISA detection method for ETX. On the one hand, it can minimize the safety hazards of the natural toxicity of ETX and the risk of amino acid back-mutation of the single-amino acid mutant (rETX m4 ). On the other hand, this method also has the characteristics of simple sample operation, low cost, rapid reaction, and strong specificity. Therefore, it can not only provide a reference for the diagnosis of Clostridium perfringens disease but also provide a basis for the screening of animals negative for Clostridium perfringens antibodies and the research on alternative methods for testing the efficacy of related vaccines. Brief Description of the Drawings

[0035] Figure 1 For the SDS-PAGE purification of rETX m1 . Detailed Embodiments

[0036] The following further describes the detailed embodiments of the present invention. It should be noted here that the description of these embodiments is for helping to understand the present invention but does not constitute a limitation on the present invention. In addition, the technical features involved in the following described embodiments can be combined with each other as long as they do not conflict with each other.

[0037] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the test materials used in the following examples are all commercially available through conventional channels unless otherwise specified.

[0038] The recombinant ETX (rETX m1 ) in this application is a non-toxic protein mutant of ETX with a single amino acid site (histidine at position 106 mutated to proline).

[0039] The ETX mutant (rETX m4 ) in this application is a four-amino acid mutant obtained by mutating the amino acids at positions 30 (tyrosine mutated to alanine), 106 (histidine mutated to proline), 196 (tyrosine mutated to alanine), and 199 (phenylalanine mutated to glutamate) of ETX.

[0040] Example 1 Antigen Preparation

[0041] 1. Natural ETX Toxin

[0042] Inoculate the Clostridium perfringens type D strain C60-2 preserved in the laboratory into anaerobic liver broth (Veterinary Biological Products Regulations of the People's Republic of China (2000 Edition)) for primary seed expansion. Then, inoculate it into liver, stomach enzyme digestion broth at an inoculation amount of 1%. After anaerobic culture, centrifuge the culture and filter to remove bacteria to obtain the natural toxin of Clostridium perfringens type D. Inactivate it with 0.8% formaldehyde solution for 7 days to obtain the inactivated natural ETX antigen.

[0043] 2. rETX m1 and rETX m4

[0044] Use the recombinant expression rETX obtained by the research team of the present invention m1 and rETX m4 expression bacteria, and perform expression, identification and purification according to the conventional method (Du Jige, Xue Qi, Zhu Zhen, et al. Expression and immunoprotective evaluation of Clostridium perfringens alpha toxin mutants [J]. Acta Veterinaria Sinica, 2019, 39(02): 265-270), and obtain rETX with a purity of more than 90% respectively m1 ( Figure 1 ) and rETX m4 two non-toxic ETX mutants.

[0045] Example 2 Screening of Clostridium perfringens ETX monoclonal antibody hybridoma cell lines with neutralizing activity and preparation of monoclonal antibody ascites

[0046] 1. Immunization of BALB / c mice and determination of antibody titer

[0047] Use the inactivated natural ETX as the immunogen to immunize female BALB / c mice by subcutaneous multi-point injection. Immunize once every 14 days. 7-10 days after the third immunization, collect an appropriate amount of blood from the orbital cavity, and use the indirect ELISA method to determine the serum titer. If the serum titer meets the fusion requirement (titer higher than 6400), then boost the immunization three days before fusion, and directly inject rETX m1 by intraperitoneal injection. Take the spleen cells of the mouse with the highest titer (1:12800) 3-5 days after the last immunization for cell fusion.

[0048] 2. Cell fusion and screening of hybridoma cell lines

[0049] Take the myeloma cell line SP2 / 0 with good growth state and the splenocytes of the mouse with the highest ELISA titer mentioned above, and carry out chemical fusion using PEG1450. Screen positive hybridoma cells by the indirect ELISA method with recombinant ETX as the coating antigen. At the same time, carry out serum neutralization experiments on the cell supernatants of the positive hybridoma cells with the natural toxin of Clostridium perfringens, and after 3 - 5 subclonings by the limiting dilution method, obtain 2 hybridoma cell lines of Clostridium perfringens ETX with neutralizing activity at the cell level (CETX-ZH1, CETX-ZH) and 1 hybridoma cell line without neutralizing activity (CETX-NH).

[0050] Table 1 Serum neutralization experiment of hybridoma cell lines

[0051]

[0052] As can be seen from Table 1, CETX-ZH with the strongest neutralizing activity is 3 times that of CETX-ZH1 with the weakest neutralizing activity, and CETX-NH does not have neutralizing activity.

[0053] Name the hybridoma cell line with the strongest neutralizing ability at the cell level as SP20 / E (i.e., CETX-ZH), and name the hybridoma cell line without neutralizing activity as SP20 / 16 (i.e., CETX-NH). And provide biological preservation of the 2 hybridoma cell lines of Clostridium perfringens ETX to the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (CGMCC) respectively. The preservation numbers are CGMCC NO.46042 (SP20 / E) and CGMCC NO.46043 (SP20 / 16). Use a subclass identification kit to detect these hybridoma cell lines and determine their subtype as IgG1. According to the regulations of the third part of the Chinese Veterinary Pharmacopoeia (2020 Edition), take the cell culture supernatant and neutralize it with natural ETX toxin, and then inject it into mice via the tail vein.

[0054] 100% (5 / 5) of the mice injected with the neutralized product of the cell supernatant of hybridoma cell CETX-ZH and the toxin survived, while all the mice injected with the neutralized product of the control cell supernatant and the toxin died, indicating that the cell supernatant of CETX-ZH also has neutralizing activity in in vivo experiments.

[0055] 3. Preparation of ascites of ETX monoclonal antibody with neutralizing activity

[0056] Select healthy multiparous BALB / c mice at 6 - 8 months old, sensitize them by intraperitoneal injection of Freund's incomplete adjuvant, and inject hybridoma cells 7 days later. Collect the above-mentioned hybridoma cells CETX-ZH in the logarithmic growth phase and SP2 / 0 cells, centrifuge, wash the cells with DMEM culture medium and then resuspend them, and adjust the cell density to 10 6After that, the mice were intraperitoneally injected. 7 to 14 days later, when the abdomen of the mice was significantly distended, a sterile needle was inserted into the distended area around the lower abdomen of the mice to allow the ascites to flow out or drip out; the ascites was collected, centrifuged, labeled as the ascites of AETX-ZH and the ascites control (S) of myeloma cells SP2 / 0, and stored frozen for later use.

[0057] Example 3 Purification and Detection of Monoclonal Ascites

[0058] The purification method using Protein A (GE Healthcare 17-5079-01) affinity column was selected for purification, and the purification steps were as follows:

[0059] Sample pretreatment: Dilute the ascites with coupling buffer (20 mM sodium phosphate buffer, pH 7.0) at a ratio of 1:3. After centrifugation, remove fat, cell residues and small particulate matter.

[0060] Equilibration: Equilibrate the column with 5 - 10 column volumes of coupling buffer, maintaining a flow rate of 2 s / drop.

[0061] Loading: Inject the sample into the upper interface of the column using a syringe, collect the effluent, and maintain a flow rate of 4 s / drop.

[0062] Washing: Pass 5 column volumes of coupling buffer through the column, maintaining a flow rate of 2 s / drop.

[0063] Elution: Elute the antibody with 5 column volumes of elution buffer (0.1 M sodium citrate buffer, pH 9.0), collect, and maintain a flow rate of 4 s / drop.

[0064] Detection of the sample: Identify the purified monoclonal antibody by SDS-PAGE.

[0065] The monoclonal antibody with a purity of 91% was obtained by the method of purification using Protein A affinity column; the concentration of the monoclonal antibody was determined by the BCA method, and its concentration could reach 3.2 mg / mL.

[0066] Example 4 Characterization of Monoclonal Antibody

[0067] (1) Dilute the purified rETX m1 and rETX m4 to 2 μg / mL with PBS, block overnight, wash with PBST, and add PBST solution containing 5% skim milk powder as the blocking solution for blocking;

[0068] (2) Take out the coated enzyme-linked immunosorbent assay (ELISA) plate, add the purified monoclonal antibody AETX-ZH, select PBS as the diluent, and dilute it 2-fold from 1:2000 to 1:128000, and incubate;

[0069] (3) Wash with PBST solution, add HRP-labeled rabbit anti-mouse antibody (secondary antibody, diluted 1:8000), and incubate.

[0070] (4) Wash with PBST solution and pat dry the enzyme-linked immunosorbent assay (ELISA) plate.

[0071] (5) Add soluble TMB substrate chromogenic solution, react in the dark at room temperature, terminate the reaction with 2M H2SO4 solution, and read the absorbance value at 450 nm.

[0072] The results are shown in Table 2. The purified monoclonal antibody AETX-ZH m1 and rETX m4 both showed good reactions, and the ELISA titers could reach 1:32000 or higher, indicating that the obtained monoclonal antibody had a high affinity for ETX.

[0073] Table 2: Detection results of the affinity of monoclonal antibody AETX-ZH

[0074] Coating protein type Concentration ELISA titer of monoclonal antibody AETX-ZH <![CDATA[rETX m1 > 2 μg / mL 1:64000 <![CDATA[rETX m4 > 2 μg / mL 1:32000

[0075] Example 5 HRP labeling of monoclonal antibody

[0076] (1) Mix antibody and activated HRP: Take 0.1 mg of antibody, add 25 μL of Reagent B, and mix well.

[0077] (2) Add 2 μL of coupling catalyst solution A to the mixture containing antibody and HRP enzyme above, and mix well.

[0078] (3) Incubate at room temperature or overnight at 4°C. The coupled antibody can be used immediately or stored at low temperature. The obtained labeled monoclonal antibody is named H-AETX-ZH.

[0079] Example 6 Establishment of a blocking ELISA detection method for Clostridium perfringens ETX antibody based on monoclonal antibody AETX-ZH

[0080] 1. Preparation of polyclonal antibody against Clostridium perfringens ETX

[0081] Using inactivated natural ETX as the immunogen, immunize sheep subcutaneously once every 14 days. Seven to ten days after the third immunization, collect blood and separate the serum.

[0082] 2. Determination of conditions for a blocking ELISA detection method for Clostridium perfringens ETX antibody

[0083] Screening of the optimal working concentrations of murine-derived polyclonal antibody and monoclonal antibody against ETX:

[0084] (1) Use PBS to dilute the purified rETX m4Dilute to 2 μg / mL, 100 μL per well, block overnight at 4°C, wash with PBST, add PBST solution containing 5% non-fat milk powder as the blocking solution, 200 μL per well, block at 37°C for 2 h;

[0085] (2) Wash with PBST solution, pat dry the enzyme-linked immunosorbent assay (ELISA) plate, and store frozen for later use;

[0086] (3) Take out the coated ELISA plate, place it at room temperature for 30 min, add the obtained polyclonal antibody against Clostridium perfringens ETX, use PBS as the diluent, and the dilution ratios are 1:100, 1:500, 1:1000, 1:2000, and 1:4000 in sequence, 100 μL per well. At the same time, add the corresponding dilution of negative serum and incubate at 37°C for 1 h;

[0087] (4) Wash with PBST solution, add the purified monoclonal antibody H-AETX-ZH labeled with horseradish peroxidase (HRP), use PBS as the diluent, dilute from 1:500 in a 2-fold dilution to 1:16000, 100 μL per well, and incubate at 37°C for 30 min;

[0088] (5) Wash with PBST solution, pat dry the ELISA plate;

[0089] (6) Finally, add 50 μL of soluble TMB substrate chromogenic solution per well, react in the dark at room temperature for 10 min, terminate the reaction with 2 M H2SO4 solution, and read the absorbance value at 450 nm.

[0090] Select the dilution ratio corresponding to the OD value of the negative control well being close to 1.0 and the blocking rate of the positive serum being ≥ 50% as the optimal working concentration.

[0091] The results showed that when the working dilution ratios of the negative serum and the polyclonal antibody against ETX were 1:500, and the working concentration of the monoclonal antibody (H-AETX-ZH) labeled with HRP was 1:8000, the OD value of the negative control well was closest to 1.0, and the blocking rate was the highest (about 80%). Therefore, the working dilution ratio of the polyclonal antibody against ETX was set at 1:500, and the working concentration of H-AETX-ZH was set at 1:8000.

[0092] Example 7 Performance Detection of the Blocking ELISA Detection Method

[0093] 1. Specificity Detection

[0094] Select a sample plate, including sera such as sheep anti-natural ETX toxin, Clostridium septicum CSA toxin, Clostridium tetani toxin, recombinant CPB toxin, and CPA toxin, and detect the ETX antibody serum by the blocking ELISA detection method.

[0095] The results showed that the detection results had good reproducibility, and there was no cross-reaction between the ETX antibody serum and other toxin antibody sera, demonstrating that the established blocking ELISA detection method had excellent specificity.

[0096] 2. Sensitivity comparison with the serum neutralization method

[0097] According to the serum neutralization method specified in the "Pharmacopoeia of the People's Republic of China" (Volume III, 2015 Edition), the sheep anti-ETX toxin polyclonal antibody prepared in Example 6 was detected by the serum neutralization method. The specific detection method was as follows:

[0098] Take 0.2 mL of rabbit anti-α toxin polyclonal antibody and mix it with 0.4 mL of natural toxin (containing different mouse MLDs, etc.). After reacting at 37 °C for 40 min, then intravenously inject 2 mice weighing 16 - 20 g, 0.3 mL / mouse. At the same time, set a challenge control and observe for 72 h.

[0099] The results showed that the neutralization titer of the sheep anti-ETX toxin polyclonal antibody against the natural toxin ETX could reach 1000 mouse MLDs / 0.1 mL.

[0100] According to the established antibody blocking ELISA operation procedure, the sheep anti-ETX toxin polyclonal antibody was diluted, and the dilution ratios were 1:500, 1:1000, 1:2000, and 1:4000 in sequence. At the same time, a negative serum control was set. After the reaction was completed, the OD 450 value was measured.

[0101] The results showed that the detection dilution of this method for ETX could reach 1:4000 and above, and the corresponding lowest detection neutralization titer could be 0.25 MLD / 0.1 mL, which was significantly lower than that of the serum neutralization method, and the sensitivity was 4 times that of the serum neutralization method.

[0102] 3. Detection of clinical samples

[0103] The blocking ELISA detection method provided by the present invention was used to detect 100 sheep sera collected clinically. The number of positive samples detected by the ETX antibody blocking ELISA method was 36, and the number of positive samples detected by the traditional serum neutralization method was 12, indicating that the ETX antibody blocking ELISA method provided by the present invention was more suitable for the detection of clinical samples.

Claims

1. A hybridoma cell line secreting monoclonal antibodies against Clostridium perfringens ETX, wherein the hybridoma cell line has a preservation number of CGMCC NO. 46042, and the monoclonal antibodies secreted by the cell line have neutralizing activity.

2. A screening method for the hybridoma cell line according to claim 1, the screening method comprising the following steps: S1. Immunize mice with natural ETX, measure the serum titer by ELISA, and use the spleen cells of the mouse with the highest titer for cell fusion; S2. Fuse the mouse spleen cells screened in S1 with myeloma cells; S3. Screen for positive clones and subclones using recombinant ETX to obtain a hybridoma cell line capable of secreting monoclonal antibodies against Clostridium perfringens ETX with neutralizing activity.

3. The method according to claim 2, in step S1, the natural ETX refers to inactivated natural ETX of Clostridium perfringens; In step S2, the myeloma cells refer to SP2 / 0 cells; In step S3, the recombinant ETX is a non-toxic single amino acid site protein mutant of ETX. The single amino acid site protein mutant is obtained by mutating the histidine at the 106th position of ETX into proline, and is named rETX m1 ; In step S3, the positive clone screening refers to screening positive hybridoma cells by an indirect ELISA method using rETX m1 as the coating antigen; the subclone screening refers to performing a serum neutralization experiment on the cell supernatant of the positive hybridoma cells with the prepared Clostridium perfringens natural toxin, and screening hybridoma cell lines with neutralizing activity after subcloning.

4. An antibody against Clostridium perfringens ETX, the antibody being a monoclonal antibody or a polyclonal antibody; the monoclonal antibody is obtained from the culture solution of the hybridoma cell line according to claim 1, or obtained by inoculating the hybridoma cell line according to claim 1 into the peritoneal cavity of a mouse to produce ascites; the polyclonal antibody is the serum obtained by immunizing mice with inactivated natural ETX and then collecting blood and centrifuging.

5. An antibody-blocking ELISA detection method for Clostridium perfringens ETX, wherein the coated antigen selected in the method is the ETX four-amino acid mutant rETX m4 , the primary antibody is the polyclonal antibody described in claim 4, and the secondary antibody is a monoclonal antibody labeled with HRP; the monoclonal antibody labeled with HRP is a monoclonal antibody obtained by purifying the ascites of the monoclonal antibody described in claim 4 and then performing HRP conjugation labeling.

6. The detection method according to claim 5, the four - amino - acid mutant rETX of ETX m4 , is a mutant rETX of ETX obtained by mutating tyrosine at position 30 of ETX to alanine, histidine at position 106 to proline, tyrosine at position 196 to alanine, and phenylalanine at position 199 to glutamic acid m4 .

7. The detection method according to claim 5, wherein the working dilution of the ETX polyclonal antibody is 1:500, and the working concentration of the HRP-conjugated monoclonal antibody is 1:8000.

8. Use of the hybridoma cell according to claim 1 or the antibody according to claim 4 in the preparation of a reagent for detecting Clostridium perfringens ETX.

9. A kit for detecting Clostridium perfringens ETX, the kit comprising the antibody according to claim 4.

10. The kit according to claim 9, wherein the kit can be one or more of an ELISA detection kit, an immunohistochemistry kit, an immunofluorescence kit, and / or an in situ hybridization staining kit.

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