Clostridium perfringens ETX hybridoma cell strain and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a hybridoma cell strain secreting a clostridium perfringens ETX monoclonal antibody 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 invention belongs to the field of biotechnology, and specifically relates to a hybridoma cell strain secreting Clostridium perfringens ETX monoclonal antibody and application thereof. Background Art

[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 an aerobic environment and low concentrations of superoxide. It has a wide range of spread and great harm (Kiu R, Hall LJ. An update on the human and animal enteric pathogen Clostridium perfringens [J]. Emerg Microbes Infect, 2018, 7 (1): 141.). The bacteria are thick rod-shaped, with straight edges, blunt ends, and even ends. They have no flagella and cannot move. They exist alone or in pairs, occasionally in short chains. They can produce spores and have capsules. As a pathogen in animals, the ability to produce capsules is one of the characteristics that distinguishes it from other clostridia. The propagation temperature range is wide (35℃-37℃), and the optimal pH value for growth is 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 a medium rich in carbon sources, the colonies are off-white, with a smooth surface and edges, a slightly concave center, and a foul odor. The spores of this species have extremely strong tolerance to external environmental conditions, can survive extreme conditions such as dryness and high temperature, and re-germinate and grow under suitable conditions. In addition, the cell wall of Clostridium perfringens contains more acetic acid, a structure that helps it survive in anaerobic environments. In an anaerobic environment, Clostridium perfringens can use organic matter for heterotrophic growth, producing a large amount of gas and metabolites; it also produces 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. ANecrotizing 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 a variety of environments, including soil, sewage and food, and is also an important component of the gastrointestinal microbiota of humans and animals in diseased or healthy states.Clostridium perfringens can cause a variety of diseases in humans and animals, including gas gangrene, food poisoning and diarrhea. According to statistics from relevant agencies, Clostridium perfringens is the main cause of food poisoning in many countries, and the incidence rate shows a trend of high incidence in autumn and winter with seasonal changes (Dolan GP, ​​Foster K, Lawler J, et al. An epidemiological review of gastrointestinal outbreaks associated with Clostridium perfringens, North East of England, 2012-2014 [J]. Epidemiol Infect. 2016 May; 144 (7): 1386-93.). In addition to causing human infection and endangering public health safety, Clostridium perfringens can also cause infection in a variety of livestock and poultry such as pigs, chickens, cattle, sheep, and quails, with the main symptoms being necrotic enteritis, diarrhea, and enterotoxemia, causing huge economic losses to the livestock and poultry 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, and toxin typing depends on whether it carries 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 type D strains. It is an inactive protoxin that is activated after proteases remove the N-terminal and C-terminal peptides (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 can cause rapid and fatal enterotoxemia in ruminant livestock such as sheep, goats and cattle, causing huge losses to the livestock industry every year. When injected into rats, the toxin accumulates mainly in the kidneys and brain, causing damage to the blood-brain barrier and possibly death. ETX is the third most potent biotoxin, second only to botulinum neurotoxin and tetanus toxin, with a 50% lethal dose (LD50) for mice. 50 ) is 65-110ng / kg, so it is classified as a Class B biological agent by the 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, causing serious economic losses to the animal husbandry industry. 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.

[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 comprises 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] In a second aspect, the present invention provides a method for screening a hybridoma cell line that secretes an anti-Clostridium perfringens ETX monoclonal antibody, the screening method comprising the following steps:

[0010] S1. Immunize mice with natural ETX, determine serum titer by ELISA, and use spleen cells of mice with the highest titer for cell fusion;

[0011] S2. Take the mouse spleen cells screened in S1 and fuse them with myeloma cells;

[0012] S3. Use recombinant ETX to screen 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 Clostridium perfringens natural ETX.

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

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

[0016] 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 ).

[0017] 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.

[0018] 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 neutralizing activity and 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] In a third aspect, the present invention provides an anti-Clostridium perfringens ETX monoclonal 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.

[0021] In a fourth aspect, the present invention provides an antigen detection method for Clostridium perfringens ETX toxin, wherein the coated 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 neutralization activity is secreted by the hybridoma cell line without neutralization activity described in the first aspect.

[0023] 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.

[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] In a fifth aspect, the present invention provides a use of the hybridoma cell 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 include Clostridium perfringens ETX monoclonal antibodies with neutralizing activity and Clostridium perfringens ETX monoclonal antibodies without neutralizing activity.

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

[0029] Furthermore, the monoclonal antibody without neutralizing activity is secreted by the hybridoma cell line without neutralizing activity described in the first aspect.

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

[0031] 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.

[0032] 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.

[0033] 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.

[0034] Beneficial Effects

[0035] 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:

[0036] (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;

[0037] (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;

[0038] (3) Using the natural toxin of Clostridium perfringens to conduct monoclonal antibody cell neutralization tests and mouse serum neutralization tests, the monoclonal antibody hybridoma cell lines with neutralization resistance and without neutralization activity against natural ETX were distinguished.

[0039] 2. Obtain a hybridoma cell line that secretes a monoclonal antibody against Clostridium perfringens ETX that is resistant to neutralization and a hybridoma cell line that is not resistant to neutralization. The above two hybridoma cell lines can stably and efficiently secrete a monoclonal antibody with neutralizing activity and a monoclonal antibody without neutralization resistance, respectively, and can achieve large-scale batch production.

[0040] 3. For the first time, an ETX antigen ELISA detection method was established using two types of ETX monoclonal antibodies with neutralizing activity and one without neutralizing activity. This method has the characteristics of simple sample operation, low cost, rapid reaction, and strong specificity. It can also provide a reference for the diagnosis of Clostridium perfringens disease and lay a foundation for the research of alternative methods for testing the efficacy of related vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 For rETX m1 Purification by SDS-PAGE. DETAILED DESCRIPTION

[0042] 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.

[0043] 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.

[0044] 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).

[0045] Example 1 Antigen preparation

[0046] 1. Natural ETX toxins

[0047] 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.

[0048] 2. rETX m1

[0049] Using the recombinant expression rETX obtained by the present invention team m1 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 the non-toxic ETX mutant rETX with a purity of more than 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 of BALB / c mice and determination of antibody titer

[0052] Female BALB / c mice were immunized with inactivated natural ETX as the immunogen by multiple subcutaneous injections. Immunization was performed 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), the immunization was strengthened three days before the fusion, and rETXm1 was directly injected intraperitoneally. 3-5 days after the last immunization, the spleen cells of the mouse with the highest titer (1:12800) were taken for cell fusion.

[0053] 2. Cell fusion and screening of hybridoma cell lines

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

[0055] Table 1 Hybridoma cell line serum neutralization experiment

[0056]

[0057]

[0058] As shown in Table 1, CETX-ZH with the strongest neutralizing activity is 3 times that of CETX-ZH1 with the weakest neutralizing activity, and CETX-NH has no neutralizing activity.

[0059] The hybridoma cell line with the strongest neutralization ability at the cellular level was named SP20 / E (i.e., CETX-ZH), and the hybridoma cell line without neutralization activity was named SP20 / 16 (i.e., CETX-NH). The two hybridoma cell lines of Clostridium perfringens ETX were provided to the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration, and the deposit numbers were CGMCC NO.46042 (SP20 / E) and CGMCC NO.46043 (SP20 / 16), respectively. The above two hybridoma cell lines were tested using a subclass identification kit, and it was determined that their subtypes were both IgG1. According to the provisions of the third volume of the "Pharmacopoeia of the People's Republic of China" (2020 edition), the cell culture supernatant was neutralized with the natural ETX toxin and then injected into mice through the tail vein.

[0060] 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.

[0061] 3. Preparation of monoclonal antibody ascites

[0062] 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 hybridoma cells CETX-ZH, hybridoma cells CETX-NH, and SP2 / 0 cells in logarithmic growth phase were collected, centrifuged, washed with DMEM culture medium, and resuspended to adjust the cell density to 10 6 After 7 to 14 days, when the abdomen of the mouse is obviously swollen, use a sterile needle to pierce the swollen part of the lower abdomen of the mouse to make the ascites flow out or drip out; collect the ascites, centrifuge, mark as AETX-ZH, AETX-NH, and freeze for later use.

[0063] Example 3 Purification and detection of monoclonal ascites

[0064] Protein A (GE Healthcare 17-5079-01) affinity column purification method was used for purification, and the purification steps were as follows:

[0065] 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.

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

[0067] 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.

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

[0069] 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.

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

[0071] 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 2.6 mg / mL.

[0072] Example 4 Characterization of Monoclonal Antibodies

[0073] (1) Purified rETX was added to PBS m1 2 μg / mL, blocked overnight, washed with PBST, and added PBST solution containing 5% skim milk powder as blocking solution, and blocked;

[0074] (2) Take out the coated ELISA plate, add purified monoclonal antibodies AETX-ZH and AETX-NH to different coated wells, use PBS as diluent, dilute the concentration by 2 times from 1:2000 to 1:128000, and 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 ELISA plate dry;

[0077] (5) Finally, add soluble TMB substrate colorimetric solution and react at room temperature in the dark. Terminate the reaction with 2M H2SO4 solution and read the absorbance at 450 nm.

[0078] The results showed that the purified monoclonal antibodies AETX-ZH and AETX-NH could bind to rETX m1 A good reaction occurred, and the ELISA titer could reach 1:32000 or above, indicating that the two monoclonal antibodies obtained had a high affinity to ETX.

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

[0080] (1) Mixing the 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, and mix well;

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

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

[0084] (1) The purified monoclonal antibody AETX-NH was coated on an ELISA plate with PBS at 4°C overnight at four coating concentrations: 25 ng / well, 50 ng / well, 100 ng / well, and 150 ng / well;

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

[0086] (3) Take out the coated ELISA plate, 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 diluent, dilute 2-fold from 1:500 to 1:16000, 100 μL / well, incubate at 37°C for 30 min;

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

[0088] (5) 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.

[0089] According to the test results of OD450 nm The P / N value was calculated and the optimal monoclonal antibody coating concentration was determined to be 100 ng / well, and the optimal HRP-coated monoclonal antibody detection dilution was determined to be 1:4000.

[0090] Example 7 Performance test of an ELISA method for detecting ETX toxin of Clostridium perfringens

[0091] 1. Specificity analysis

[0092] Sample plates were selected, including natural ETX toxin, Clostridium putrefaciens CSA toxin, Clostridium tetani toxin, recombinant CPB toxin, CPA toxin, etc., and the ETX toxin sample plates were tested by the ELISA test method.

[0093] The results showed that the test results had good reproducibility and there was no cross-reaction between natural ETX toxin and other toxins, proving that the prepared test strips had excellent specificity.

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

[0095] According to the previous data, the obtained ETX natural toxin was diluted in different multiples to obtain toxins of different concentrations, and the mouse toxicity test and ELISA detection methods of ETX were performed respectively.

[0096] The results showed that the sensitivity of an ELISA detection method for Clostridium perfringens ETX toxin could be as low as 6 times that of the mouse challenge method.

[0097] 3. Evaluation of vaccine products

[0098] Six multi-dry powder vaccines containing ETX toxins were tested using an ELISA method for Clostridium perfringens ETX toxin, and the detection rate of ETX toxin was 100%. However, since the ETX components of currently commercialized Clostridium vaccines are all inactivated non-toxic toxins, it is impossible to determine the toxicity using mice, indicating that this method can provide a material basis for antigen detection of Clostridium vaccines.

Claims

1. A hybridoma cell line secreting an anti-Clostridium perfringens ETX monoclonal antibody, the hybridoma cell line comprising CGMCC NO.46042 and CGMCC NO.46043; the monoclonal antibody secreted by the hybridoma cell line CGMCC NO.46042 has neutralizing activity, and the monoclonal antibody secreted by the hybridoma cell line CGMCC NO.46043 has no 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 Clostridium perfringens ETX monoclonal antibodies.

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 screening hybridoma cell lines with neutralization activity and without neutralization activity after subcloning; 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.

4. An anti-Clostridium perfringens ETX monoclonal 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.

5. A method for detecting an antigen of 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.

6. The detection method according to claim 5, wherein the Clostridium perfringens ETX monoclonal antibody without neutralization activity is secreted by the hybridoma cell line without neutralization activity according to claim 1; and 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 it with HRP.

7. The detection method according to claim 5, wherein the coating dilution of the monoclonal antibody without neutralizing activity is 100 ng / well; and the detection dilution of the HRP-labeled monoclonal antibody is 1:4000.

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

9. A kit for detecting Clostridium perfringens ETX, the kit comprising a monoclonal antibody against Clostridium perfringens ETX; the monoclonal antibodies include a monoclonal antibody against Clostridium perfringens ETX with neutralizing activity and a monoclonal antibody against Clostridium perfringens ETX without neutralizing activity; the monoclonal antibody with neutralizing activity is secreted by the hybridoma cell line with neutralizing activity according to claim 1; the monoclonal antibody without neutralizing activity is secreted by the hybridoma cell line without neutralizing activity according to claim 1.

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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