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 selecting natural toxins, an indirect ELISA detection method is established by utilizing nontoxic ETX single amino acid site protein mutants, a series of hybridoma cell strains secreting anti-clostridium perfringens ETX monoclonal antibodies are screened, and the hybridoma cell strains can stably and efficiently secrete the monoclonal antibodies with neutralizing activity. According to the present invention, the clostridium perfringens ETX monoclonal antibody with neutralization resistance is combined with the ETX tetraamino acid mutant to establish the ETX antibody blocking ELISA detection method, and the method can reduce the potential safety hazard of the ETX natural toxicity and the potential safety hazard of the single amino acid mutant amino acid reverse mutation; the kit also has the characteristics of simplicity and convenience in operation, low cost, quick reaction, strong specificity and the like, and can provide reference for diagnosis of clostridium perfringens disease.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a hybridoma cell line of Clostridium perfringens ETX monoclonal antibody secreting neutralizing activity and application thereof. 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 a variety of systemic and gastrointestinal diseases in humans and animals, including gas gangrene, food poisoning, gastrointestinal diseases, and liver and kidney damage in humans, 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 from A to G" (ROOD JI, ADAMS V, LACEY J, et al. Expansion of the Clostridium perfringens toxin-based typing scheme[J].Anaerobe.2018Oct;53:5-10.). Type A Clostridium perfringens mainly causes animal enterotoxemia, gas gangrene and necrotizing enteritis, etc.; Type B, C, D and E Clostridium perfringens mainly cause lamb dysentery, sheep dysentery, enterotoxemia and other diseases. Type C can also cause necrotizing enteritis in humans; Type F can cause foodborne diseases in humans and lead to watery diarrhea; Type G is closely related to necrotizing enteritis in chickens (Xu Jirong, Zhao Cui, Fan Li, et al. Analysis of drug resistance and molecular genetic evolution characteristics of Clostridium perfringens 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 D. It is the third most potent Clostridial toxin after botulinum and tetanus neurotoxins, and the most potent toxin among all exotoxins of Clostridium perfringens. The toxin is 296 amino acids long and is secreted outside the bacteria in the form of a toxin precursor (HUNTER SE, 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 upon by the host's trypsin, chymotrypsin or Clostridium's own protease, 11 to 13 amino acids at the N-terminus and 22 to 29 amino acids at the C-terminus are removed, thereby activating the mature toxin (FREEDMAN JC, MCCLANE BA, UZALF A. New insights into Clostridium perfringens epsilon toxin activation and action on the brain during enterotoxemia [J]. Anaerobe, 2016, 41: 27-31.). The toxin belongs to the pore-forming toxin family and is mainly divided into three regions, I, II and III, in terms of structure. They play an important role in the process of toxin binding to cell receptors, the maintenance of the stability of binding to cell receptors and the formation of cell membrane perforations. Since the toxin has two peptide chains that span the three regions I, II and III at the same time, the strategy of using only one domain of the toxin as a candidate vaccine antigen (such as the C-terminus of α and β toxins) is difficult to apply to the prevention and control of the toxin (Du Jige, Peng Xiaobing, Zhang Xiukun, et al. Expression and immune protection evaluation of Clostridium perfringens ε toxin mutants [J]. Chinese Journal of Veterinary Drugs, 2018, 52(6): 13-20.). As a potential biological weapon, the toxicity of ETX is second only to botulinum toxin and tetanus toxin. It can cause severe enterotoxemia in poultry and livestock, especially ruminants, causing serious economic losses to animal husbandry. Therefore, achieving attenuation or even non-toxicity of ETX while retaining its immunogenicity, and then preparing appropriate vaccines and other biological preparations, is of great significance in terms of both economy and biosafety. The soluble recombinant ETX obtained by Li Qing et al. through the prokaryotic system has strong toxicity, which leads to the fact that the wild-type recombinant ETX also has biosafety risks such as toxin leakage or incomplete inactivation during the preparation of the toxin subunit vaccine (Li Qing. Construction and application of mutants of α and ε toxins of Clostridium perfringens [D]. Anhui: Anhui Medical University, 2013.).

[0004] At present, traditional natural toxin inactivated vaccines are usually used for immunization prevention for animals such as cattle, sheep and rabbits, but there is a lack of relevant toxin detection and treatment methods. Due to the complex antigen composition and low effective content of natural toxin inactivated vaccines, it is easy to cause side effects in animals, resulting in the inability to be used for prevention of animals such as dogs and pigs. Existing studies have shown that the preparation of monoclonal antibodies can provide a basis for the establishment of treatment and diagnosis of certain toxins. The key to the preparation of monoclonal antibodies is the immunogen and the coating antigen, but the preparation, purification and inactivation of natural toxins are complicated and have high biosafety risks. Summary of the invention

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

[0006] In a first aspect, the present invention provides a hybridoma cell line secreting an anti-Clostridium perfringens ETX monoclonal antibody, wherein the hybridoma cell line is selected from any one of the accession numbers CGMCC NO.46042 and CGMCC NO.46043, and the monoclonal antibody secreted by the cell line has neutralizing activity.

[0007] In a second aspect, the present invention provides a method for screening the hybridoma cell line according to the first aspect, the screening method comprising the following steps:

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

[0009] S2. Take the mouse spleen cells screened in S1 and fuse them with myeloma cells;

[0010] S3. Use recombinant ETX to screen positive clones and subclones to obtain hybridoma cell lines that can secrete anti-Clostridium perfringens ETX monoclonal antibodies with neutralizing activity.

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

[0012] Furthermore, in step S2, the myeloma cells are SP2 / 0 cells.

[0013] Furthermore, in step S2, the fusion method is a 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 is mutated to proline) protein mutant (rETX m1 ).

[0015] Further, in step S3, the positive clone screening refers to the use of rETX m1 The positive hybridoma cells were screened by indirect ELISA method using coated antigen.

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

[0017] In a third aspect, the present invention provides an anti-Clostridium perfringens ETX antibody, which is a monoclonal antibody or a polyclonal antibody; the monoclonal antibody is obtained from the culture medium of the hybridoma cell line described in the first aspect, or is obtained by inoculating the hybridoma cell line described in the first aspect into the peritoneal cavity of mice to produce ascites; the polyclonal antibody is obtained by immunizing mice with inactivated natural ETX and then collecting blood to obtain serum after centrifugation.

[0018] In a fourth aspect, the present invention provides an antibody blocking ELISA detection method for Clostridium perfringens ETX, wherein the coating antigen is selected from 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] Furthermore, the four amino acid mutants of ETX, rETX m4 The ETX mutant (rETX) was obtained by mutating the amino acids at positions 30 (tyrosine to alanine), 106 (histidine to proline), 196 (tyrosine to alanine), and 199 (phenylalanine to glutamate) of ETX. m4 ).

[0020] Furthermore, the HRP-labeled monoclonal antibody is a monoclonal antibody obtained by purifying the ascites of the monoclonal antibody described in the third aspect and then coupling and labeling it with HRP.

[0021] Furthermore, the working dilution of the ETX polyclonal antibody is 1:500, and the working concentration of the HRP-conjugated monoclonal antibody is 1:8000.

[0022] In a fifth aspect, the present invention provides a use of the hybridoma cell described in the first aspect or the antibody described in the third aspect in preparing a reagent for detecting Clostridium perfringens ETX.

[0023] In a sixth aspect, the present invention provides a kit for detecting Clostridium perfringens ETX, wherein the kit comprises the antibody according to the third aspect.

[0024] Furthermore, the kit may 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 method of the kit includes 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 anti-ETX monoclonal antibodies with the accession number of CGMCC NO.46042 in the present invention has been provided with biological deposit to the General Microbiology Center (CGMCC) of China Microorganism Culture Collection Administration Committee. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The classification name is: mouse hybridoma cell, and the deposit date is August 19, 2024.

[0027] The hybridoma cell line secreting anti-ETX monoclonal antibodies with the accession number of CGMCC NO.46043 in the present invention has been provided with biological deposit to the General Microbiology Center (CGMCC) of China Microorganism Culture Collection Administration Committee. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The classification name is: mouse hybridoma cell, and the deposit date is August 19, 2024.

[0028] Beneficial Effects

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

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

[0031] (2) An ELISA detection method was established using a non-toxic ETX mutant with a purity of more than 90% as a coating antigen, and positive clones and subclones were screened, which not only ensured the similarity between the coating antigen and the natural toxin, but also avoided the cumbersome purification steps and biosafety risks of the natural ETX of Clostridium perfringens;

[0032] (3) Using the natural toxin of Clostridium perfringens to conduct a monoclonal antibody neutralization test in cells and a mouse serum neutralization test, a monoclonal antibody hybridoma cell line with neutralization resistance to natural ETX was obtained.

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

[0034] 3. For the first time, a monoclonal antibody with neutralization resistance to ETX from Clostridium perfringens was used to bind to a four-amino acid mutant of ETX (rETX m4 ) established an antibody blocking ELISA assay for ETX, which can minimize the potential safety risks of ETX's natural toxicity and single amino acid mutants (rETX m4 ) On the other hand, this method has the characteristics of simple sample operation, low cost, rapid reaction and strong specificity, which can not only provide a reference for the diagnosis of Clostridium perfringens disease, but also provide a basis for the screening of Clostridium perfringens antibody-negative animals and the research on alternative methods for related vaccine efficacy testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 For rETX m1 SDS-PAGE purification.

[0036] Figure 2 SDS-PAGE identification of rETXm4. Note: M, Marker; 1, 0.5 mg / ml BSA; 2, uninduced control (BL21); 3-5: IPTG induced (BL21). DETAILED DESCRIPTION

[0037] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0039] In this application, recombinant ETX (rETX m1 ) is a non-toxic ETX protein mutant with a single amino acid site (histidine at position 106 mutated to proline).

[0040] In this application, ETX mutant (rETX m4) are four amino acid mutants 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.

[0041] Example 1 Antigen preparation

[0042] 1. Natural ETX toxins

[0043] The D-type Clostridium perfringens C60-2 strain preserved in the laboratory was inoculated into anaerobic meat liver soup (Regulations of the People's Republic of China on Veterinary Biological Products) (2000 edition) for primary seed propagation, and then inoculated into meat liver gastric enzyme digestion soup at an inoculum of 1%. After anaerobic culture, the culture was centrifuged and filtered for sterilization to obtain the D-type Clostridium perfringens natural toxin. The natural ETX antigen was obtained by inactivation with 0.8% formaldehyde solution for 7 days.

[0044] 2. rETX m1 and rETX m4

[0045] Using the recombinant expression rETX obtained by the present invention team m1 and rETX m4 The expression bacteria were expressed, identified and purified according to conventional methods (Du Jige, Xue Qi, Zhu Zhen, et al. Expression and immune protection evaluation of α-toxin mutants of Clostridium perfringens [J]. Chinese Journal of Veterinary Medicine, 2019, 39(02): 265-270), and rETX with a purity of more than 90% were obtained. m1 ( Figure 1 ) and rETX m4 ( Figure 2 ) Two avirulent ETX mutants.

[0046] Example 2 Screening of hybridoma cell lines secreting neutralizing Clostridium perfringens ETX monoclonal antibodies and preparation of monoclonal antibody ascites

[0047] 1. Immunization of BALB / c mice and determination of antibody titer

[0048] Female BALB / c mice were immunized with inactivated natural ETX as the immunogen by multiple subcutaneous injections. Immunization was performed once every 14 days. 7-10 days after the third immunization, an appropriate amount of blood was collected from the eye sockets and the serum titer was determined by indirect ELISA. If the serum titer reached the fusion requirement (the titer was higher than 6400), booster immunization was performed three days before fusion, and rETX was directly used. m1 Intraperitoneal injection. 3-5 days after the last immunization, spleen cells from mice with the highest titer (1:12800) were used for cell fusion.

[0049] 2. Cell fusion and screening of hybridoma cell lines

[0050] Myeloma cells SP2 / 0 with good growth status were chemically fused with the spleen cells of mice with the highest titer in the above ELISA test using PEG1450, and positive hybridoma cells were screened by indirect ELISA method with recombinant ETX as the coating antigen. At the same time, the cell supernatant of positive hybridoma cells was subjected to serum neutralization experiment with the prepared natural toxin of Clostridium perfringens. After 3 to 5 subclonings by limiting dilution method, 2 hybridoma cell lines of Clostridium perfringens ETX with neutralizing activity at the cellular level and 1 hybridoma cell line without neutralizing activity were obtained. The hybridoma cell line with the strongest neutralizing ability at the cellular level was named CETX-ZH. This hybridoma cell line was detected by subclass identification kit, and its subtype was determined to be IgG1. According to the provisions of the third part of the "Pharmacopoeia of the People's Republic of China" (2020 edition), the cell culture supernatant was neutralized with natural ETX toxin and injected into mice through the tail vein.

[0051] 100% (5 / 5) of the mice injected with the cell supernatant of hybridoma cells CETX-ZH and the toxin neutralizer survived, while all the mice injected with the control cell supernatant and the toxin neutralizer died, indicating that the CETX-ZH cell supernatant also has neutralizing activity in the in vivo experiment.

[0052] 3. Preparation of ascites of ETX monoclonal antibody with neutralizing activity

[0053] Healthy multiparous BALB / c mice aged 6 to 8 months were selected and sensitized by intraperitoneal injection of Freund's incomplete adjuvant. Hybridoma cells were injected 7 days later. The above-mentioned logarithmic growth phase hybridoma cells CETX-ZH and SP2 / 0 cells were collected, centrifuged, washed with DMEM culture medium, and resuspended. The cell density was adjusted to 10 6 After 7 to 14 days, when the abdomen of the mouse is obviously swollen, a needle is aseptically inserted into the swollen part of the lower abdomen of the mouse to make the ascites flow out or drip out; the ascites is collected, centrifuged, and marked as AETX-ZH and ascites control (S) of myeloma cells SP2 / 0, and frozen for later use.

[0054] Example 3 Purification and detection of monoclonal ascites

[0055] Protein A (GE Healthcare 17-5079-01) affinity column purification method was used for purification, and the purification steps were as follows:

[0056] Sample pretreatment: Ascites was diluted 1:3 with coupling buffer (20 mM sodium phosphate buffer, pH 7.0), and fat, cell residues and small particulate matter were removed after centrifugation.

[0057] Equilibration: Equilibrate the column with 5-10 column volumes of coupling buffer, maintaining a flow rate of 2 s / drop.

[0058] Loading: Use a syringe to inject the sample into the upper interface of the column, collect the effluent, and maintain a flow rate of 4s / drop.

[0059] Washing: Pass 5 column volumes of coupling buffer through the column, maintaining a flow rate of 2s / drop.

[0060] Elution: Elute the antibody with 5 column volumes of elution buffer (0.1 M sodium citrate buffer, pH 9.0), collect, and maintain the flow rate at 4 s / drop.

[0061] Sample detection: The purified monoclonal antibody was identified by SDS-PAGE.

[0062] The monoclonal antibody with a purity of 91% was obtained by purification using Protein A affinity column; the concentration of the monoclonal antibody was determined by BCA method, and its concentration could reach 3.2 mg / mL.

[0063] Example 4 Characterization of Monoclonal Antibodies

[0064] (1) Purified rETX was added to PBS m1 and rETX m4 All were diluted to 2 μg / mL, blocked overnight, washed with PBST, and blocked by adding PBST solution containing 5% skim milk powder;

[0065] (2) Take out the coated ELISA plate, add the purified monoclonal antibody AETX-ZH, use PBS as the diluent, dilute the concentration by 2 times from 1:2000 to 1:128000, and incubate;

[0066] (3) Wash with PBST solution, add HRP-labeled rabbit anti-mouse antibody (secondary antibody, 1:8000 dilution) and incubate;

[0067] (4) Wash with PBST solution and pat the ELISA plate dry;

[0068] (5) Add soluble TMB substrate colorimetric solution, react at room temperature in the dark, terminate the reaction with 2M H2SO4 solution, and read the absorbance at 450 nm.

[0069] The results are shown in Table 1. The purified monoclonal antibody AETX-ZH and rETX m1 and rETX m4 All of them could react well, and the ELISA titer could reach 1:32000 or above, indicating that the obtained monoclonal antibodies had a high affinity for ETX.

[0070] Table 1: Affinity test results of monoclonal antibody AETX-ZH

[0071] 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

[0072] Example 5 HRP labeling of monoclonal antibodies

[0073] (1) Mixing the antibody and activated HRP: Take 0.1 mg of antibody, add 25 μL of reagent B, and mix well;

[0074] (2) Add 2 μL of coupling catalyst solution A to the mixture of antibody and HRP enzyme, and mix well;

[0075] (3) Allow the reaction to stand 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.

[0076] Example 6 Establishment of a blocking ELISA detection method for Clostridium perfringens ETX antibody based on monoclonal antibody AETX-ZH

[0077] 1. Preparation of anti-Clostridium perfringens ETX polyclonal antibodies

[0078] Sheep were immunized subcutaneously with inactivated natural ETX as the immunogen once every 14 days. Blood was collected and serum was separated 7 to 10 days after the third immunization.

[0079] 2. Determination of the conditions for a blocking ELISA detection method for Clostridium perfringens ETX antibodies

[0080] Screening of optimal working concentrations of mouse ETX polyclonal and monoclonal antibodies:

[0081] (1) Purified rETX was added to PBS m4 Dilute to 2 μg / mL, 100 μL / well, block overnight at 4°C, wash with PBST, add PBST solution containing 5% skim milk powder as blocking solution, 200 μL / well, block at 37°C for 2 h;

[0082] (2) Wash with PBST solution, pat the ELISA plate dry, and freeze for later use;

[0083] (3) Take out the coated ELISA plate, place it at room temperature for 30 min, add the obtained anti-Clostridium perfringens ETX polyclonal antibody, use PBS as the dilution solution, the dilution ratio is 1:100, 1:500, 1:1000, 1:2000 and 1:4000, 100 μL / well, add the negative serum of the corresponding dilution ratio, and incubate at 37°C for 1 h;

[0084] (4) Wash with PBST solution, add purified HRP-labeled monoclonal antibody H-AETX-ZH, use PBS as diluent, dilute 2-fold from 1:500 to 1:16000, 100 μL / well, incubate at 37°C for 30 min;

[0085] (5) Wash with PBST solution and pat the ELISA plate dry;

[0086] (6) Finally, add 50 μL / well of soluble TMB substrate colorimetric solution and react for 10 min at room temperature in the dark. Terminate the reaction with 2M H2SO4 solution and read the absorbance at 450 nm.

[0087] The OD value of the negative control well is selected to be close to 1.0, and the dilution corresponding to the positive serum blocking rate ≥50% is the optimal working concentration.

[0088] The results showed that when the working dilution of negative serum and ETX polyclonal antibody was 1:500, and the working concentration of HRP-labeled monoclonal antibody (H-AETX-ZH) 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 of ETX polyclonal antibody was set at 1:500, and the working concentration of H-AETX-ZH was set at 1:8000.

[0089] Example 7 Blocking ELISA Detection Method Performance Test

[0090] 1. Specificity detection

[0091] A sample plate was selected, including sheep sera against natural ETX toxin, Clostridium putrefaciens CSA toxin, Clostridium tetani toxin, recombinant CPB toxin, CPA toxin, etc., and the ETX antibody sera were tested by blocking ELISA detection method.

[0092] The results showed that the test results had good reproducibility, and there was no cross-reaction between ETX antibody sera and other toxin antibody sera, proving that the established blocking ELISA detection method had excellent specificity.

[0093] 2. Comparison of sensitivity with serum neutralization method

[0094] According to the serum neutralization method specified in the Veterinary Pharmacopoeia of the People's Republic of China (Volume III, 2015 edition), the sheep anti-ETX toxin polyclonal antibody prepared in Example 6 was tested by the serum neutralization method. The specific detection method is:

[0095] Take 0.2mL of rabbit anti-α toxin polyclonal antibody and mix it with 0.4mL of natural toxin (containing different mouse MLD, etc.), incubate at 37℃ for 40min, then inject intravenously into 2 mice weighing 16-20g, 0.3mL / mouse. At the same time, set up a challenge control and observe for 72h.

[0096] The results showed that the neutralization titer of the sheep anti-ETX toxin polyclonal antibody against the natural toxin ETX could reach 1000 mouse MLD / 0.1mL.

[0097] According to the established antibody blocking ELISA operating procedure, the sheep anti-ETX toxin polyclonal antibody was diluted to 1:500, 1:1000, 1:2000 and 1:4000, and a negative serum control was set. After the reaction was completed, the OD 450 value.

[0098] The results showed that the dilution of ETX detection by this method could reach 1:4000 and above, and the corresponding minimum detection neutralization titer could be 0.25MLD / 0.1mL, which was significantly lower than the serum neutralization method and its sensitivity was 4 times that of the serum neutralization method.

[0099] 3. Testing of clinical samples

[0100] 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 is more suitable for the detection of clinical samples.

Claims

1. A hybridoma cell line secreting an anti-Clostridium perfringens ETX monoclonal antibody, wherein the hybridoma cell line is selected from any one of the deposit numbers CGMCC NO.46042 or CGMCC NO.46043, and the monoclonal antibody secreted by the cell line has neutralizing activity.

2. A method for screening a hybridoma cell line according to claim 1, comprising the following steps: S1. Immunize mice with natural ETX, determine serum titer by ELISA, and use spleen cells of mice with the highest titer for cell fusion; S2. Take the mouse spleen cells screened in S1 and fuse them with myeloma cells; S3. Use recombinant ETX to screen positive clones and subclones to obtain hybridoma cell lines that can secrete anti-Clostridium perfringens ETX monoclonal antibodies with neutralizing activity.

3. The method according to claim 2, wherein in step S1, the natural ETX refers to the inactivated natural ETX of Clostridium perfringens; In step S2, the myeloma cells are SP2 / 0 cells; In step S3, the recombinant ETX is a non-toxic ETX single amino acid site protein mutant, wherein the single amino acid site protein mutant is a mutant in which the histidine at position 106 of ETX is mutated to proline, and is named rETX m1 . In step S3, the positive clone screening refers to the use of rETX m1 The indirect ELISA method of coated antigen is used to screen positive hybridoma cells; the subclone screening refers to using the prepared natural toxin of Clostridium perfringens to conduct serum neutralization experiments on the cell supernatant of positive hybridoma cells, and then screening hybridoma cell lines with neutralization activity after subcloning.

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

5. An antibody blocking ELISA detection method for Clostridium perfringens ETX, wherein the coating antigen is selected from the ETX four amino acid mutant rETX m4 The primary antibody is the polyclonal antibody according to claim 4, and the secondary antibody is an HRP-labeled monoclonal antibody; the HRP-labeled monoclonal antibody is a monoclonal antibody obtained by purifying the ascites of the monoclonal antibody according to claim 4 and then coupling and labeling it with HRP.

6. The detection method according to claim 5, wherein the four amino acid mutants of ETX are rETX m4 , is an ETX mutant rETX obtained by mutating the tyrosine at position 30 to alanine, the histidine at position 106 to proline, the tyrosine at position 196 to alanine, and the phenylalanine at position 199 to glutamate. 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 preparing a reagent for detecting Clostridium perfringens ETX.

9. A kit for detecting Clostridium perfringens ETX, 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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