A Clostridium perfringens ETX hybridoma cell line and its application

Abstract

This invention belongs to the field of biotechnology, specifically relating to a hybridoma cell line secreting monoclonal antibodies against Clostridium perfringens ETX and its applications. Based on natural toxins, this invention establishes an indirect ELISA detection method using a non-toxic ETX single-amino acid site protein mutant. A series of hybridoma cell lines secreting monoclonal antibodies against Clostridium perfringens ETX were screened. These hybridoma cell lines can stably and efficiently secrete both neutralizing monoclonal antibodies and non-neutralizing monoclonal antibodies, and can be mass-produced on a large scale. This invention is the first to establish an antigen ELISA detection method for Clostridium perfringens ETX using monoclonal antibodies. This method features simple sample handling, low cost, rapid reaction, and high specificity. It can also provide a reference for the diagnosis of Clostridium perfringens infection and lay the foundation for research on alternative methods for testing the efficacy of related vaccines.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a hybridoma cell line that secretes Clostridium perfringens ETX monoclonal antibody and its application. Background Technology

[0002] Clostridium perfringens (CP), also known as Clostridium perfringens, is an important zoonotic Gram-positive anaerobic bacterium. Although it is an anaerobic bacterium, it can still survive in aerobic environments and under low concentrations of superoxide, resulting in a wide spread and significant harm (Kiu R, Hall LJ. An update on the human and animalenteric pathogen Clostridium perfringens[J]. Emerg Microbes Infect, 2018, 7(1):141.). The bacterium is rod-shaped with straight edges, blunt and rounded ends, and relatively even apex. It lacks flagella and is non-motile. It exists singly or in pairs, occasionally in short chains. It can produce spores and has a capsule. As a pathogen in animals, the ability to produce a capsule is one of the characteristics that distinguishes it from other clostridiums. The bacteria reproduce over a wide temperature range (35℃-37℃), with an optimal growth pH of 6.5 to 7.5 (DaveG AA rapid qualitative assay for detection of Clostridium perfringens incanned food products[J].Acta Biochim Pol,2017,64(2):207-213.). In carbon-rich media, the colonies are grayish-white, smooth with edges, slightly concave in the center, and emit a foul odor. The spores of this strain are highly tolerant of external environmental conditions, surviving under extreme conditions such as dryness and high temperature, and re-germinating and growing under suitable conditions. In addition, the cell wall of Clostridium perfringens contains a large amount of acetic acid, a structure that helps it survive in anaerobic environments. In anaerobic environments, *Clostridium perfringens* can utilize organic matter for heterotrophic growth, producing large amounts of gas and metabolic products. It also forms a capsule, which makes it more stable against the external environment and increases its ability to colonize and invade the host (HEIDA FH, VAN ZOONEN AGJF, HULSCHER JBF, et al. *Anecrotic Enterocolitis-Associated Gut Microbiota Is Present in the Meconium: Results of a Prospective Study*[J]. *Clinical Infectious Diseases*, 2016, 62(7):863-70.). *Clostridium perfringens* exists in various environments, including soil, sewage, and food, and is an important component of the gastrointestinal microbiota in both sick and healthy humans and animals.Clostridium perfringens can cause a variety of diseases in humans and animals, including gas gangrene, food poisoning, and diarrhea. According to statistics from relevant organizations, Clostridium perfringens is a major cause of food poisoning in many countries, and the incidence rate shows a trend of high incidence in autumn and winter (Dolan GP, ​​Foster K, Lawler J, et al. An epidemiological review of gas intestinal outbreaks associated with Clostridium perfringens, North East of England, 2012-2014[J]. Epidemiol Infect. 2016 May;144(7):1386-93.). Besides causing human infection and endangering public health, Clostridium perfringens can also cause infection in various livestock and poultry, such as pigs, chickens, cattle, sheep, and quails, with the main symptoms being necrotic enteritis, diarrhea, and enterotoxemia. This has caused huge economic losses to the livestock and poultry farming industry worldwide (Alimolaei M, Ezatkhah M, Soltani S. Toxin genotypes of Clostridium perfringens isolates from common quail (Coturnix coturnix) with or without acute necroticenteritis[J]. Toxicon. 2023 Jan 1; 221:106984; Kalender H, ). H, Timurkaan N, etal. Detection and molecular characterization of Clostridium perfringens, Paeniclostridium sordellii and Clostridium septicum from lambs and goat kids with hemorrhagic abomasitis in Turkey[J]. BMC Vet Res. 2023 Jan 13; 19(1):8.).

[0003] Clostridium perfringens can secrete more than 20 identified toxins. Toxin typing depends on the presence of the cpa / plc, cpb, etx, iap / ibp / itx, cpe, and netB genes, which encode six toxins: α, β, ε, ι, CPE, and NetB, respectively (Yao PY, Annamaraju P. Clostridium perfringens Infection[J].2024). Epsilon-Toxin (ETX) is encoded by the etx gene plasmid of Clostridium perfringens type B and D strains. It is an inactive prototoxin that is activated by the removal of the N- and C-terminal peptides by proteases (MINAMI J, KATAYAMA S, MATSUSHITA O, et al. Lambda-Toxin of Clostridium perfringens Activates the Precursor of Epsilon-Toxin by Releasing Its N- and C-Terminal Peptides[J]. Microbiology and Immunology, 2013, 41(7): 527-35.). ETX causes rapid and fatal enterotoxemia in ruminant livestock such as sheep, goats, and cattle, resulting in huge losses to the livestock industry every year. When injected into rats, the toxin mainly accumulates in the kidneys and brain, causing damage to the blood-brain barrier and potentially death. ETX is the third most potent biotoxin, after botulinum neurotoxin and tetanus toxin, with a 50% lethal dose (LD50) in mice. 50 The concentration is 65-110 ng / kg, therefore it is classified as a Category B biological agent by the US Centers for Disease Control and Prevention (ALVES GG, MACHADO DE). RA,CH VEZ-OL RTEGUI CD, et al. Clostridium perfringens epsilon toxin: The third most potent bacterial toxin known[J]. Anaerobe, 2014, 30: 102-7.). ETX can cause severe enterotoxemia in poultry and livestock, especially ruminants, resulting in serious economic losses to the livestock industry. Therefore, achieving attenuation or even non-toxicity of ETX while preserving its immunogenicity, and then preparing appropriate vaccines and other biological agents, is of great significance in terms of both economics and biosafety.

[0004] Currently, traditional inactivated natural toxin vaccines are commonly used for immunization of animals such as cattle, sheep, and rabbits, but related toxin detection and treatment methods are lacking. Because inactivated natural toxin vaccines have complex antigenic components and low effective content, they are prone to causing adverse reactions in animals, making them unsuitable for prevention in animals such as dogs and pigs. Existing research indicates that the preparation of monoclonal antibodies can provide a foundation for the treatment and diagnosis of certain toxins. The key to monoclonal antibody preparation is the immunogen and coating antigen, but the preparation, purification, and inactivation processes of natural toxins are complex and pose significant biosafety risks. Summary of the Invention

[0005] This invention, based on the selection of natural toxins, utilizes a non-toxic ETX single-amino acid site mutant (histidine at position 106 is mutated to proline) protein (rETX). m1 An indirect ELISA detection method was established, and a series of hybridoma cell lines secreting monoclonal antibodies against Clostridium perfringens ETX 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 that secretes monoclonal antibodies against Clostridium perfringens ETX, the hybridoma cell line comprising CGMCC NO.46042 and CGMCC NO.46043.

[0007] Furthermore, the monoclonal antibody secreted by the hybridoma cell line CGMCC NO.46042 has neutralizing activity.

[0008] Furthermore, the monoclonal antibody secreted by the hybridoma cell line CGMCC NO.46043 has no neutralizing activity.

[0009] Secondly, the present invention provides a method for screening hybridoma cell lines that secrete monoclonal antibodies against Clostridium perfringens ETX, the screening method comprising the following steps:

[0010] S1. Mice were immunized with natural ETX, and serum titers were determined by ELISA. Spleen cells from mice with the highest titers were used for cell fusion.

[0011] S2. Fuse mouse spleen cells selected in S1 with myeloma cells;

[0012] S3. Use recombinant ETX to screen for positive clones and subclones to obtain hybridoma cell lines that can secrete Clostridium perfringens ETX monoclonal antibodies.

[0013] Furthermore, in step S1, the natural ETX refers to the inactivated natural ETX of Clostridium perfringens.

[0014] Furthermore, in step S2, the myeloma cells refer to SP2 / 0 cells.

[0015] Furthermore, in step S2, the fusion method is a PEG-mediated chemical method.

[0016] Furthermore, in step S3, the recombinant ETX is a non-toxic ETX single-amino acid site mutant (histidine at position 106 is mutated to proline) protein mutant (rETX). m1 ).

[0017] Furthermore, in step S3, the positive clone screening refers to using rETX m1 To screen positive hybridoma cells using an indirect ELISA method coated with antigen.

[0018] Furthermore, in step S3, the subcloning screening refers to performing a serum neutralization experiment on the cell supernatant of positive hybridoma cells using the prepared Clostridium perfringens natural toxin, and then screening hybridoma cell lines with neutralizing activity and those without neutralizing activity after subcloning.

[0019] Furthermore, in step S3, the hybridoma cell lines obtained by screening include hybridoma cell lines that secrete Clostridium perfringens ETX monoclonal antibodies with neutralizing activity and hybridoma cell lines that secrete Clostridium perfringens ETX monoclonal antibodies without neutralizing activity.

[0020] Thirdly, the present invention provides a monoclonal antibody against Clostridium perfringens ETX; the monoclonal antibody is obtained from the culture medium 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 peritoneal cavity of mice to produce ascites.

[0021] Fourthly, the present invention provides an antigen detection method for Clostridium perfringens ETX toxin, wherein the coating antibody is a Clostridium perfringens ETX monoclonal antibody without neutralizing activity, and the detection antibody is an HRP-labeled Clostridium perfringens ETX monoclonal antibody with neutralizing activity.

[0022] Furthermore, the Clostridium perfringens ETX monoclonal antibody without neutralizing activity is secreted by the hybridoma cell line without neutralizing activity described in the first aspect.

[0023] Furthermore, the HRP-labeled monoclonal antibody is a monoclonal antibody that is HRP-conjugated after purifying the ascites fluid of the monoclonal antibody described in the third aspect.

[0024] Furthermore, the coating dilution of the monoclonal antibody without neutralizing activity is 100 ng / well.

[0025] Furthermore, the detection dilution of the HRP-labeled monoclonal antibody is 1:4000.

[0026] Fifthly, the present invention provides the use of the hybridoma cells described in the first aspect or the monoclonal antibody described in the third aspect in the preparation of a reagent for detecting Clostridium perfringens ETX.

[0027] In a sixth aspect, the present invention provides a kit for detecting Clostridium perfringens ETX, the kit comprising Clostridium perfringens ETX monoclonal antibodies; the monoclonal antibodies comprising Clostridium perfringens ETX monoclonal antibodies with neutralizing activity and Clostridium perfringens ETX monoclonal antibodies without neutralizing activity.

[0028] Furthermore, the neutralizing monoclonal antibody is secreted by the neutralizing hybridoma cell line described in the first aspect.

[0029] Furthermore, the non-neutralizing monoclonal antibody is secreted by the non-neutralizing hybridoma cell line described in the first aspect.

[0030] Furthermore, the kit may be one or more of the following: ELISA detection kit, immunohistochemistry kit, immunofluorescence kit, and / or in situ hybridization staining kit.

[0031] Furthermore, the diagnostic methods of the kit include one or more of the following: direct method, indirect method, double-antibody sandwich method, and / or competitive method.

[0032] The hybridoma cell line secreting anti-ETX monoclonal antibody, with accession number CGMCC NO.46042, has been deposited with the China General Microbiological Culture Collection Center (CGMCC) at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The cell line is classified as mouse hybridoma cell and the deposit date is August 19, 2024.

[0033] The hybridoma cell line secreting anti-ETX monoclonal antibody, with accession number CGMCC NO.46043, has been deposited with the China General Microbiological Culture Collection Center (CGMCC) at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The cell line is classified as mouse hybridoma cell and the deposit date is August 19, 2024.

[0034] Beneficial effects

[0035] 1. This invention is the first to utilize natural ETX as an immunogenic antigen, a non-toxic monoamino acid ETX mutant as a coating antigen, and combines a cell neutralization assay for Clostridium perfringens natural toxin and a mouse serum neutralization assay to screen hybridoma cell lines with neutralizing monoclonal antibodies. The implementation of the above method has the following advantages:

[0036] (1) Using a small amount of natural ETX as an immunogen ensures that the natural conformation of ETX is maintained to the greatest extent.

[0037] (2) An ELISA detection method was established using a non-toxic ETX mutant with a purity of over 90% as the coating antigen, and positive clones and subclones were screened to ensure the similarity between the coating antigen and the natural toxin, while avoiding the cumbersome purification steps and biosafety hazards of Clostridium perfringens natural ETX.

[0038] (3) Using Clostridium perfringens natural toxin to conduct cell neutralization test and mouse serum neutralization test for monoclonal antibodies, thereby distinguishing monoclonal antibody hybridoma cell lines with neutralizing resistance to natural ETX and those without neutralizing activity.

[0039] 2. Obtain hybridoma cell lines of Clostridium perfringens ETX monoclonal antibody that secrete neutralizing antibodies and those without neutralizing antibodies. The two hybridoma cell lines can stably and efficiently secrete neutralizing monoclonal antibodies and those without neutralizing antibodies, respectively, and can be mass-produced on a large scale.

[0040] 3. For the first time, an antigen ELISA detection method for Clostridium perfringens ETX was established using two monoclonal antibodies with neutralizing and non-neutralizing activities. This method is characterized by simple sample handling, low cost, rapid reaction, and high specificity. It can also provide a reference for the diagnosis of Clostridium perfringens infection and lay the foundation for the research of alternative methods for testing the efficacy of related vaccines. Attached Figure Description

[0041] Figure 1 For rETX m1 SDS-PAGE purification. Detailed Implementation

[0042] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, 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.

[0043] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0044] In this application, ETX (rETX) is reorganized. m1 It is a non-toxic ETX single amino acid site mutant (histidine at position 106 is mutated to proline).

[0045] Example 1 Antigen Preparation

[0046] 1. Natural ETX toxin

[0047] The laboratory-preserved Clostridium perfringens strain C60-2 was inoculated into anaerobic meat and liver broth (Regulations for Veterinary Biological Products of the People's Republic of China) (2000 edition) for primary seed propagation. Then, a 1% inoculum was added to the meat and liver digestion broth. After anaerobic culture, the culture was centrifuged and filtered for sterilization to obtain the natural toxin of Clostridium perfringens type D. This toxin was then inactivated using 0.8% formaldehyde solution for 7 days to obtain the inactivated natural ETX antigen.

[0048] 2. rETX m1

[0049] Using the recombinant expression rETX obtained by the team of this invention m1 The expression strain was expressed, identified, and purified using conventional methods (Du Jige, Xue Qi, Zhu Zhen, et al. Expression and immunoprotective efficacy evaluation of Clostridium perfringens α-toxin mutant [J]. Chinese Journal of Veterinary Medicine, 2019, 39(02):265-270), and a non-toxic ETX mutant rETX with a purity of over 90% was obtained. m1 ( Figure 1 ).

[0050] Example 2: Screening of monoclonal antibody hybridoma cell lines and preparation of monoclonal antibody ascites.

[0051] 1. Immunization and antibody titer determination in BALB / c mice

[0052] Female BALB / c mice were immunized subcutaneously at multiple sites using inactivated natural ETX as the immunogen. Immunization was repeated every 14 days. Seven to ten days after the third immunization, blood was collected from the orbital sinus, and serum titer was determined using an indirect ELISA method. If the serum titer met the fusion requirements (titer higher than 6400), a booster immunization was administered three days before fusion, via intraperitoneal injection of rETXm1. Spleen cells from mice with the highest titer (1:12800) were collected 3-5 days after the final immunization for cell fusion.

[0053] 2. Screening of cell fusion and hybridoma cell lines

[0054] Myeloma cells SP2 / 0 in good growth condition were chemically fused with mouse spleen cells that showed the highest titer in the above ELISA test using PEG1450. Positive hybridoma cells were screened using an indirect ELISA method with recombinant ETX as the coating antigen. At the same time, the cell supernatant of the positive hybridoma cells was subjected to a cell-level serum neutralization experiment using the prepared Clostridium perfringens natural toxin. After 3 to 5 subclonings using the limiting dilution method, two Clostridium perfringens ETX hybridoma cell lines with neutralizing activity at the cell level (CETX-ZH1, CETX-ZH) and one hybridoma cell line without neutralizing activity (CETX-NH) were obtained.

[0055] Table 1. Serum neutralization experiment of hybridoma cell lines

[0056]

[0057]

[0058] As shown in Table 1, CETX-ZH, which has the strongest neutralizing activity, is three times more active than CETX-ZH1, which has the weakest neutralizing activity, while CETX-NH has no neutralizing activity.

[0059] The hybridoma cell line with the strongest neutralizing capacity at the cellular level was named SP20 / E (CETX-ZH), and the hybridoma cell line with no neutralizing activity was named SP20 / 16 (CETX-NH). Both Clostridium perfringens ETX hybridoma cell lines were submitted to the China General Microbiological Culture Collection Center (CGMCC) for biopreservation, with accession numbers CGMCC NO.46042 (SP20 / E) and CGMCC NO.46043 (SP20 / 16), respectively. Using a subtype identification kit, both hybridoma cell lines were confirmed to be IgG1. Following the provisions of the Pharmacopoeia of the People's Republic of China (2020 Edition), Volume III, the cell culture supernatant was neutralized with natural ETX toxin and then injected into mice via the tail vein.

[0060] Mice injected with the supernatant of hybridoma cells CETX-ZH and the toxin neutralizer had 100% (5 / 5) survival, while mice injected with the control cell supernatant and the toxin neutralizer all died, indicating that the CETX-ZH cell supernatant also has neutralizing activity in vivo.

[0061] 3. Preparation of monoclonal antibody ascites

[0062] Healthy, multiparous BALB / c mice aged 6–8 months were selected and sensitized with Freund's incomplete adjuvant via intraperitoneal injection. Seven days later, hybridoma cells were injected. Hybridoma cells in the logarithmic growth phase (CETX-ZH, CETX-NH, and SP2 / 0 cells) were collected, centrifuged, washed with DMEM medium, resuspended, and adjusted to a cell density of 102. 6 After administering the fluid at a rate of 1 / mL, inject the fluid into the mice intraperitoneally. 7–14 days later, when the mice’s abdomen is noticeably distended, use a sterile needle to puncture the distended area in the lower abdomen of the mice to allow the ascites to flow out or drip out; collect the ascites, centrifuge, label it as AETX-ZH and AETX-NH, and freeze for later use.

[0063] Example 3: Purification and Detection of Monoclonal Ascites Fluid

[0064] The Protein A (GE Healthcare 17-5079-01) affinity column was used for purification. The purification steps are as follows:

[0065] Sample pretreatment: Dilute the ascites fluid 1:3 with coupling buffer (20mM sodium phosphate buffer, pH 7.0), centrifuge, and remove fat, cell debris and small particulate matter.

[0066] Equilibration: Equilibrate the column with 5-10 column volumes of coupling buffer at a flow rate of 2 s / drop.

[0067] Sample loading: Inject the sample into the upper interface of the column using a syringe, collect the eluent, and maintain a flow rate of 4 s / drop.

[0068] Washing: Pass the column through 5 column volumes of coupling buffer at a flow rate of 2 s / drop.

[0069] Elution: Elute the antibody with 5 column volumes of elution buffer (0.1M sodium citrate buffer, pH 9.0), collect, and maintain a flow rate of 4 s / drop.

[0070] Sample detection: The purified monoclonal antibody was identified by SDS-PAGE.

[0071] Monoclonal antibodies with a purity of 91% were obtained by purification using a Protein A affinity column; the concentration of monoclonal antibodies was determined by the BCA method, and the concentration reached 2.6 mg / mL.

[0072] Example 4 Characterization of Monoclonal Antibodies

[0073] (1) Use PBS to purify rETX m1 2 μg / mL, block overnight, wash with PBST, add PBST solution containing 5% skim milk powder as blocking solution, and block;

[0074] (2) Take out the coated microplate and add the purified monoclonal antibodies AETX-ZH and AETX-NH to different coated wells respectively. Use PBS as the dilution buffer and dilute the concentration from 1:2000 to 1:128000 by 2 times. Incubate.

[0075] (3) Wash with PBST solution, add HRP-labeled rabbit anti-mouse antibody (secondary antibody, 1:8000 dilution), and incubate;

[0076] (4) Wash with PBST solution and pat the plate dry;

[0077] (5) Finally, 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 450nm.

[0078] The results showed that the purified monoclonal antibodies AETX-ZH and AETX-NH could both bind to rETX. m1 The two monoclonal antibodies obtained showed a good reaction, with ELISA titers reaching 1:32000 or higher, indicating that they have a high affinity for ETX.

[0079] Example 5: HRP labeling of monoclonal antibody AETX-ZH

[0080] (1) Mixing antibody and activated HRP: Take 0.1 mg of antibody, add 25 μL of reagent B, and mix well;

[0081] (2) Add 2 μL of coupling catalyst solution A to the mixture of antibody AETX-ZH and HRP enzyme that has been added above, and mix well;

[0082] (3) Allow the reaction to stand at room temperature for 2 hours, or react overnight at 4°C. The conjugated antibody can then be used or stored at low temperature. The resulting labeled monoclonal antibody is named H-AETX-ZH.

[0083] Example 6: Establishment of an ELISA method for detecting Clostridium perfringens ETX antigen based on monoclonal antibodies AETX-ZH and AETX-NH

[0084] (1) The purified monoclonal antibody AETX-NH was coated with PBS at four coating concentrations of 25 ng / well, 50 ng / well, 100 ng / well and 150 ng / well, respectively, and the plate was incubated overnight at 4°C.

[0085] (2) Wash with PBST solution, pat the microplate dry, and freeze for later use;

[0086] (3) Take out the coated microplate, place it at room temperature for 30 min, add natural ETX toxin and blank control, incubate for 30 min, wash with PBST solution, add purified HRP-labeled monoclonal antibody H-AETX-ZH, use PBS as dilution buffer, dilute 2 times from 1:500 to 1:16000, 100 μL / well, incubate at 37℃ for 30 min;

[0087] (4) Wash with PBST solution and pat the plate dry;

[0088] (5) Finally, add 50 μL of soluble TMB substrate colorimetric solution per well, react at room temperature in the dark for 10 min, terminate the reaction with 2M H2SO4 solution, and read the absorbance at 450 nm.

[0089] Based on the OD450 of the test results nm The P / N ratio was calculated to determine the optimal monoclonal antibody coating concentration as 100 ng / well and the optimal HRP-coated monoclonal antibody detection dilution as 1:4000.

[0090] Example 7: Performance Testing of an ELISA Method for Detecting Clostridium perfringens ETX Toxin

[0091] 1. Specificity analysis

[0092] Sample trays were selected, including natural ETX toxin, Clostridium putrefactive CSA toxin, Clostridium tetani toxin, recombinant CPB toxin, CPA toxin, etc., and the ETX toxin sample trays were detected by this ELISA detection method.

[0093] The results showed that the test strips had good reproducibility and no cross-reactivity with natural ETX toxins and other toxins, proving that the prepared test strips had excellent specificity.

[0094] 2. Comparison of sensitivity with mouse challenge method

[0095] Based on previous data, the obtained ETX natural toxin was diluted by different multiples to obtain different concentrations of toxin, and the mouse toxicity of ETX and ELISA detection methods were used to determine them.

[0096] The results showed that the sensitivity of a Clostridium perfringens ETX toxin ELISA detection method was at least 6 times that of the mouse challenge method.

[0097] 3. Evaluation of vaccine products

[0098] A novel ELISA method for detecting Clostridium perfringens ETX toxin was used to detect ETX toxoids in six multivalent dry powder vaccines, achieving a 100% detection rate. However, since the ETX components in currently commercially available Clostridium vaccines are all inactivated, non-toxic toxoids, virulence cannot be determined using mice. This indicates that this method can provide a material basis for antigen detection in Clostridium vaccines.

Claims

1.A hybridoma cell strain secreting monoclonal antibody against Clostridium perfringens epsilon toxin, wherein the hybridoma cell strain comprises CGMCC NO. 46042 and CGMCC NO. 46043; the monoclonal antibody secreted by the hybridoma cell strain CGMCC NO. 46042 has neutralizing activity, and the monoclonal antibody secreted by the hybridoma cell strain CGMCC NO. 46043 has no neutralizing activity. 2.A monoclonal antibody against Clostridium perfringens epsilon toxin, wherein the monoclonal antibody is obtained from the culture solution of the hybridoma cell strain of claim 1, or is obtained by inoculating the hybridoma cell strain of claim 1 into the abdominal cavity of a mouse to produce ascites. 3.Use of the hybridoma cell of claim 1 or the monoclonal antibody of claim 2 in the preparation of a reagent for detecting Clostridium perfringens epsilon toxin. 4.A kit for detecting Clostridium perfringens epsilon toxin, wherein the kit comprises monoclonal antibodies against Clostridium perfringens epsilon toxin, wherein the monoclonal antibodies comprise monoclonal antibodies against Clostridium perfringens epsilon toxin with neutralizing activity and monoclonal antibodies against Clostridium perfringens epsilon toxin without neutralizing activity; the monoclonal antibodies with neutralizing activity are secreted by the hybridoma cell strain with neutralizing activity of claim 1; and the monoclonal antibodies without neutralizing activity are secreted by the hybridoma cell strain without neutralizing activity of claim 1. 5.The kit of claim 4, wherein the kit is selected from one or more of an ELISA detection kit, an immunohistochemical kit, an immunofluorescence kit and / or an in situ hybridization staining kit.

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