Colloidal gold detection method of clostridium perfringens ETX

Abstract

The invention belongs to the technical field of biology, and particularly relates to a colloidal gold detection method of clostridium perfringens ETX. Natural ETX is used as an immunizing antigen for the first time, a non-toxic single amino acid ETX mutant is used as a coating antigen, and a cell neutralization test of clostridium perfringens natural toxin and a serum neutralization test of mice are combined to screen the monoclonal antibody hybridoma cell strain with neutralization activity. The screened 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 colloidal gold detection method, and the method has characteristics of simple sample operation, low cost, rapid reaction, strong specificity and the like, and can provide the basis for the rapid diagnosis of clostridium perfringens disease.

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 an application thereof. Background Art

[0002] Clostridium perfringens (CP) was first isolated from a decaying corpse in 1892 and named Clostridium perfringens. CP can be widely found in natural environments such as plants, soil, rivers, incompletely processed animal products, and the gastrointestinal tract of humans and animals. It is a zoonotic pathogen that can be transmitted between humans, animals and the natural environment through multiple pathways (Derongs L, Druilhe C, Ziebal C, et al. Characterization of Clostridium Perfringens Isolates Collected from Three Agricultural Biogas Plants over a One-Year Period[J]. Int J Environ Res Public Health. 2020 Jul 29; 17(15): 5450.). Usually, these strains do not cause disease in animals. However, when the intestinal environment is damaged or the external environment changes dramatically, Clostridium perfringens proliferates in large numbers in the intestine and produces a variety of toxins at the same time, causing animal disease (Navarro MA, McClane BA, Uzal FA. Mechanisms of Action and Cell Death Associated with Clostridium perfringens Toxins [J]. Toxins (Basel). 2018 May 22; 10 (5): 212.). According to the CPA, CPB, ETX, ITX, CPE and NetB toxins produced by Clostridium perfringens, it can be divided into 7 types of toxins: A to G. Type A only produces CPA toxin; Type B produces CPA, CPB and ETX toxins; Type C produces CPA and CPB toxins; Type D produces CPA and ETX toxins; Type E produces CPA and ITX toxins; Type F produces CPA and CPE toxins; Type G produces CPA and NetB toxins (ROOD JI, ADAMS V, LACEY J, et al. Expansion of the Clostridium perfringenstoxin-based typing scheme[J]. Anaerobe. 2018 Oct; 53: 5-10.).Different toxin types of Clostridium perfringens can cause a variety of human and animal diseases. Type A can cause food poisoning in humans and enteritis in livestock and wild animals; Type B mainly causes neonatal dysentery in lambs; Type C causes sudden death in cattle and sheep, necrotic enteritis in poultry, enterotoxemia in neonatal ruminants, and red dysentery in piglets; Type D mainly causes enterotoxemia in sheep, goats and cattle; Type E can cause dysentery in calves and lambs (LIJ, ADAMS V, BANNAM TL, et al. Toxin plasmids of Clostridium perfringens [J]. Microbiol Mol Biol Rev. 2013 Jun; 77(2): 208-33.).

[0003] Epsilon toxin (ETX) is produced by type B and type D C. perfringens strains. It is encoded by the toxin gene etx located on the pCW3-like plasmid, consists of 296 amino acids, and has a molecular weight of 33 kDa (Miyata S, Matsushita O, Minami J, et al. Cleavage of a C-terminal peptide is essential for heptamerization of Clostridium perfringens epsilon-toxin in the synaptosomalmbrane [J]. J Biol Chem. 2001 Apr 27; 276 (17): 13778-83.). ETX is a key toxin that causes disease in Clostridium perfringens type D. The toxin is weak in toxicity when it is first produced. It can be activated by removing the N-terminal and C-terminal residues through various proteases in the intestine or by cleaving the connection between the 10th and 11th amino acids of the N-terminus through the λ protease produced by the bacteria themselves (Freedman JC, Li J, Uzal FA, et al. Proteolytic processing and activation of Clostridium perfringens epsilon toxin by caprine small intestinal contents [J]. mBio. 2014 Oct 21; 5(5): e01994-14.). The activated ETX is an extremely potent clostridial toxin that can act on multiple organs such as the kidneys, lungs and brain. The median lethal dose reaches 70 ng / kg, second only to botulinum toxin and tetanus toxin. The toxin can be embedded in the cell membrane to form perforations, and can also act on nerve cells to promote the release of neurotransmitters, resulting in increased vascular permeability, rupture of intestinal epithelial cells, and rupture of astrocytes around blood vessels in brain tissue, leading to various clinical symptoms such as perivascular edema, increased intracranial pressure, and local softening of brain tissue (Popoff MR. Epsilon toxin: a fascinating pore-forming toxin [J]. FEBS J. 2011 Dec; 278 (23): 4602-15.). 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. However, Li Qing et al. found that recombinant ETX still has strong toxicity. 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 the hybridoma cell line according to the first aspect, 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] Further, in step S3, the hybridoma cell lines 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 a colloidal gold detection method for Clostridium perfringens ETX toxin, wherein the monoclonal antibody with neutralizing activity is a gold-labeled antibody, and the monoclonal antibody without neutralizing activity is used as the detection antibody.

[0022] Furthermore, the monoclonal antibody with neutralizing activity is secreted by the hybridoma cell line with neutralizing activity described in the first aspect.

[0023] Furthermore, the monoclonal antibody without neutralizing activity is secreted by the hybridoma cell line without neutralizing activity described in the first aspect.

[0024] 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.

[0025] 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.

[0026] Furthermore, the monoclonal antibody with neutralizing activity is secreted by the hybridoma cell line with neutralizing activity described in the first aspect.

[0027] Furthermore, the monoclonal antibody without neutralizing activity is secreted by the hybridoma cell line without neutralizing activity described in the first aspect.

[0028] Furthermore, the kit may be one or more of a colloidal gold kit, an ELISA detection kit, an immunohistochemistry kit, an immunofluorescence kit and / or an in situ hybridization staining kit.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] Beneficial Effects

[0033] 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:

[0034] (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;

[0035] (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;

[0036] (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.

[0037] 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.

[0038] 3. For the first time, a colloidal gold detection method for ETX was established using two types of Clostridium perfringens ETX monoclonal antibodies with and without neutralization resistance. This method has the characteristics of simple sample operation, low cost, rapid reaction, and strong specificity, and can provide a basis for the rapid diagnosis of Clostridium perfringens disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 For rETX m1 SDS-PAGE purification.

[0040] Figure 2 Schematic diagram of colloidal gold test strips for ETX toxin detection. DETAILED DESCRIPTION

[0041] 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.

[0042] 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.

[0043] 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).

[0044] Example 1 Antigen preparation

[0045] 1. Natural ETX toxins

[0046] 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.

[0047] 2. rETX m1

[0048] 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 Example 2 Screening of hybridoma cell lines secreting monoclonal antibodies to Clostridium perfringens ETX with or without neutralizing activity

[0049] 1. Immunization of BALB / c mice and determination of antibody titer

[0050] 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.

[0051] 2. Cell fusion and screening of hybridoma cell lines

[0052] 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.

[0053] Table 1 Hybridoma cell line serum neutralization experiment

[0054]

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 3. Preparation of monoclonal antibody ascites

[0059] 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 106 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.

[0060] Example 3 Purification and detection of monoclonal ascites

[0061] Protein A (GE Healthcare 17-5079-01) affinity column purification method was used for purification, and the purification steps were as follows:

[0062] 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.

[0063] Equilibration: Equilibrate the column with 5-10 column volumes of coupling buffer, maintaining a flow rate of 2 s / drop.

[0064] 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.

[0065] Washing: Pass 5 column volumes of coupling buffer through the column, maintaining a flow rate of 2s / drop.

[0066] 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.

[0067] Sample detection: The purified monoclonal antibody was identified by SDS-PAGE.

[0068] Two monoclonal antibodies, AETX-ZH and AETX-NH, were obtained by purification using a Protein A affinity column with purities of 90% or more. The concentrations of the monoclonal antibodies were determined using the BCA method, and the concentrations of AETX-ZH and AETX-NH were up to 2.6 mg / mL and 2.7 mg / mL, respectively.

[0069] Example 4 Characterization of Monoclonal Antibodies

[0070] (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;

[0071] (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;

[0072] (3) Wash with PBST solution, add HRP-labeled rabbit anti-mouse antibody (secondary antibody, 1:8000 dilution) and incubate;

[0073] (4) Wash with PBST solution and pat the ELISA plate dry;

[0074] (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.

[0075] 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.

[0076] Example 5 Preparation of monoclonal antibody AETX-ZH colloidal gold-monoclonal antibody immune complex

[0077] Add 100mL of colloidal gold solution to a beaker and adjust the pH to 8.0. Add 1.6mg of AETX-ZH monoclonal antibody and stir at room temperature. Add boric acid buffer (100g / L, pH 8.0) and stir. Add 10% BSA to block and stir at room temperature. Centrifuge the labeled complex, discard the precipitate, centrifuge the supernatant again, discard the supernatant, resuspend the precipitate to the original volume with PBS (0.01M pH 8.2, containing 0.1% BSA, 0.05% NaN3), repeat the centrifugation as above, resuspend the final precipitate in a container, and store at low temperature for later use.

[0078] Example 6 Establishment of a colloidal gold detection method for Clostridium perfringens based on monoclonal antibodies AETX-ZH and AETX-NH

[0079] 1. Assembly of test strips

[0080] 1.0 μg of monoclonal antibody AETX-NH was used as the detection line (T line), and 1.5 μg of rabbit anti-mouse polyclonal antibody was coated about 0.6 cm away from the detection line as the quality control line (C line). The coating amount of colloidal gold-labeled monoclonal antibody AETX-ZH coated on the conjugate pad was 3.5 μg. The above antibodies were sprayed at different voltages and flow rates. Subsequently, the nitrocellulose membrane was dried at room temperature, placed in a desiccant, and sealed for storage. The test strip consists of a PVP plastic base, a sample pad, a colloidal gold conjugate pad, a water-absorbing filter paper, and an NC membrane. The entire assembly process must be completed in a clean, constant temperature and humidity environment. The colloidal gold labeled with AETX-ZH monoclonal antibody is adsorbed on the glass cellulose membrane as a colloidal gold conjugate pad. The NC membrane is glued to the base plate, the water-absorbing filter paper and the conjugate pad overlap at 1 cm of the NC membrane, and the sample pad overlaps at 3 cm of the conjugate pad. The sample liquid is added to the sample pad and chromatographed along the direction of the water-absorbing pad, thereby generating an immune reaction. The design and reaction principle of the test strip are as follows Figure 2 shown.

[0081] 2. Judgment conditions

[0082] In the reaction area of ​​the test strip, if red stripes appear on both the test line and the quality control line, the sample to be tested is judged to be positive; if a red stripe appears only on the quality control line, it is judged to be negative; if there is no red stripe at the quality control line, regardless of whether there is a red stripe at the test line, it means that the test strip is invalid and needs to be retested.

[0083] Example 7 Performance test of an ELISA method for detecting ETX toxin of Clostridium perfringens

[0084] 1. Specificity analysis

[0085] A sample plate was selected, including natural ETX toxin, Clostridium putrefaciens CSA toxin, Clostridium tetani toxin, recombinant CPB toxin, CPA toxin, etc., and the ETX toxin sample was tested by the immunochromatographic test strip of Example 6.

[0086] The results showed that the test results had good reproducibility and there was no cross-reaction between ETX toxin and other toxins, proving that the prepared test strip had excellent specificity.

[0087] 2. Comparison of sensitivity with mouse challenge method

[0088] 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.

[0089] The results showed that the sensitivity of an ELISA detection method for Clostridium perfringens ETX toxin was at least twice that of the mouse challenge method.

[0090] 3. Evaluation of vaccine products

[0091] 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 then screening hybridoma cell lines with neutralization activity and without neutralization activity after subcloning.

4. The method according to any one of claims 2 or 3, wherein the screened hybridoma cell lines 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.

5. 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.

6. A colloidal gold detection method for ETX toxin of Clostridium perfringens, wherein the monoclonal antibody with neutralizing activity is a gold-labeled antibody, and the monoclonal antibody without neutralizing activity is used as the detection antibody.

7. The detection method according to claim 6, wherein the monoclonal antibody with neutralizing activity is secreted by the hybridoma cell line with neutralizing activity according to claim 1; and the monoclonal antibody without neutralizing activity is secreted by the hybridoma cell line without neutralizing activity according to claim 1.

8. Use of the hybridoma cell according to claim 1 or the monoclonal antibody according to claim 5 in preparing a reagent for detecting Clostridium perfringens ETX.

9. 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; the monoclonal antibodies with neutralizing activity are secreted by the hybridoma cell line with neutralizing activity according to claim 1; the monoclonal antibodies without neutralizing activity are 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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