Competitive elisa kit for detection of bovine contagious pleuropneumonia and its application

Abstract

The application discloses a competitive ELISA detection kit for bovine contagious pleuropneumonia and application thereof. The kit contains a monoclonal antibody secreted by a hybridoma cell strain with a microbial preservation number of CGMCC NO.45502 and an antigen-coated ELISA plate composed of amino acids 29 to 166 in the extracellular region of a small colony false protein of Mycoplasma mycoides subsp. mycoides (Mmm). The application successfully screens a candidate diagnostic marker protein through a whole protein analysis method of Mycoplasma mycoides subsp. mycoides (Mmm). A detection method established by screening a monoclonal antibody with the protein can specifically detect CBPP positive serum and has no cross reaction with other related disease positive serum. The competitive ELISA detection method established by the application has a specific property equivalent to that of a WOAH recommended method and a sensitivity about 25% higher than that of the WOAH recommended method. The application provides an effective technical means for detection of bovine contagious pleuropneumonia.

Description

TECHNICAL FIELD

[0001] The present application relates to a competitive ELISA detection kit for contagious bovine pleuropneumonia and its application, and belongs to the field of biological medicine. BACKGROUND

[0002] Contagious bovine pleuropneumonia (CBPP) is a contagious animal disease of susceptible animals of the bovine family caused by Mycoplasma mycoides subsp. mycoides (Mmm), and the susceptible animals are mainly cattle, but there are also reports of Mmm isolated from sheep and goats. The incubation period of the disease is from 3 weeks to 6 months, and the disease can be divided into acute, subacute and chronic forms according to the clinical manifestations. The diseased cattle mainly show clinical symptoms such as rapid breathing, fever, runny nose, anorexia, etc., and severe cases can suffocate and die, and when the disease first occurs in the herd, the mortality rate can be more than 50%, and during the outbreak of the epidemic, the mortality rate can reach 90%. However, subacute and chronic forms are very common, and subacute and chronic forms can cause subclinical infection, which can be one of the important reasons for the continuous spread of the disease. The earliest report of CBPP can be traced back to Europe in the 16th century, and in the 16th century, the disease was limited to the Alps and Pyrenees regions in Europe, and in the 19th century, it spread rapidly across the European continent through the Netherlands and Switzerland, and in 1850, it was introduced into Scandinavia, the United States and Australia through the United Kingdom, and in the late 19th century, it was introduced into New Zealand, India, China, Mongolia, Korea and Japan through Australia. In the early 20th century, many countries eliminated CBPP through culling or culling after immunization. At present, CBPP is still prevalent in sub-Saharan Africa, and it is reported that the direct and indirect economic losses caused by CBPP in Africa each year are as high as 4480 million euros (€44.8 million). China introduced CBPP when it introduced dairy cows in 1919, and the last diseased animal was culled in 1989, and since then there has been no diseased animal, and in 2011 it became a CBPP-free country through WOAH certification. According to the WOAH report, although the disease is mainly prevalent in Africa, it cannot be determined that the countries and regions around China are free of CBPP, so China still faces the threat of the disease.

[0003] Accurate and rapid diagnosis is very important for successful control of CBPP epidemic, researchers have been working hard in the field of CBPP detection method. In the detection of pathogens, in addition to pathogen isolation and identification, polymerase chain reaction test (PCR) has become an important means of identification of Mmm, and a variety of traditional PCR detection methods and real-time PCR detection methods have been developed. Serological detection methods include complement fixation test (CFT) (Amanfu, W., Sediadie, S., Masupu, K. V., Raborokgwe, M. V., Benkirane, A., Geiger, R., and Thiaucourt, F. (2000) Comparison between c-ELISA and CFT in detecting antibodies to Mycoplasma mycoides mycoides biotype SC in cattle affected by CBPP in Botswana. Annals of the New York Academy of Sciences 916, 364-369), latex agglutination (March, J. B., Kerr, K., and Lema, B. (2003) Rapid detection of contagious bovine pleuropneumonia by a Mycoplasma mycoides subsp. mycoides SC capsular polysaccharide-specific antigen detection latex agglutination test. Clin Diagn Lab Immunol 10, 233-240), slide agglutination tests (Turner, A., and Etheridge, J. (1963) Slide agglutination tests in the diagnosis of contagious bovine pleuropneumonia.Australian Veterinary Journal 39, 445-451), Immunoblotting (IB), Competitive ELISA (C-ELISA), Indirect ELISA (i-ELISA) (Lutta, H. O., Mather, A., Maina, T. W., Odongo, D., Ndiwa, N. N., Wesonga, H. O., and Naessens, J. (2018) Preliminary Findings of Lipoprotein B in Detecting Cattle Chronically Infected with Contagious Bovine Pleuropneumonia. Journal of Veterinary Science & Medical Diagnosis 07) and Immunohistochemical (IHC) (Luciani, M., Armillotta, G., Manna, L., Ciarelli, A., Di Febo, T., Krasteva, I., Pini, A., Sacchini, F., and D'Angelo, A. R. (2016) Selection of a monoclonal antibody by ELISA, immunoblotting and Quartz Crystal Microbalance technology for immunohistochemical detection of Mycoplasma mycoides subsp. mycoides. Veterinaria italiana) among others. Among them, IB, CFT and C-ELISA are the recommended detection methods by WOAH. The sensitivity and specificity of CFT are 63.8% and 98% respectively, while the sensitivity of C-ELISA is comparable to CFT and the specificity can reach 99.9% (Amanfu, W., Sediadie, S., Masupu, K., Benkirane, A., Geiger, R., and Thiaucourt, F. (1998) Field validation of a competitive enzyme-linked immunosorbent assay (cELISA) for the detection of contagious bovine pleuropneumonia in Botswana.REVUE D'ELEVAGE ET DE MEDICINE VETERINAIRE DES PAYS TROPICAUX 51, 189-193; Le Goff, C., and Thiaucourt, F. (1998) A competitive ELISA for the specific diagnosis of contagious bovine pleuropneumonia (CBPP). Vet Microbiol 60, 179-191). But unfortunately, so far, there is no detection method that can detect all animals in different stages of the disease.

[0004] At present, China has no commercial CBPP serological detection kit with independent intellectual property rights, and the serological detection of CBPP depends on imported products to a great extent, but in the past two years, foreign kits have not been able to be purchased. Therefore, it is urgent to develop a commercial CBPP serological detection kit with independent intellectual property rights. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a competitive ELISA detection kit for contagious bovine pleuropneumonia and its application.

[0006] In order to achieve the above purpose, the following technical means are adopted in the present application:

[0007] In order to establish a competitive ELISA detection method for serum antibodies of contagious bovine pleuropneumonia, the present application analyzes all the encoded proteins of Mycoplasma mycoides subsp. Mycoides small colony strain PG1, and selects a region on a transmembrane protein as having the value of establishing a serological detection method. A monoclonal antibody 3A8 is obtained by using the protein as an immunogen. A competitive ELISA detection method is established by using the extracellular region of the protein as a coating antigen, 3A8 as a competitive antibody, and HRP-labeled goat anti-mouse IgG as an enzyme-labeled antibody. The detection method is used to detect the antibody growth and decline rule of artificially immunized sheep serum, and the established method is compared with a commercial kit. On this basis, the established detection method is applied to monitor more than 1000 sera from border provinces of China. The results show that the present application successfully selects a candidate diagnostic marker protein by analyzing the Mmm whole protein. The detection method established by using the monoclonal antibody selected from the protein can specifically detect CBPP positive serum without cross reaction with other related disease positive serum. The CBPP cELISA detection method established in the present application has a different research and development scheme from the traditional method, which can greatly reduce the investment of human and material resources, save research and development time, and provide a new idea for the research of mycoplasma and other complex pathogen serological detection methods.

[0008] On the basis of the above-mentioned research, firstly, the application provides a hybridoma cell strain secreting a mycoplasma mycoides subsp. mycoides small colony specific monoclonal antibody, and the hybridoma cell strain is named Mmm-3A8, and the classification name is a hybridoma cell secreting Mmm-3A8 monoclonal antibody, and is preserved in the General Microbiological Center of the Chinese Microorganism Bacterium Preserving Management Committee, located at No. 1, Beichen West Road, Hua-yuan District, Beijing, and the address is the Institute of Microbiology of the Chinese Academy of Sciences, and the microorganism preserving number is CGMCC NO. 45502, and the preserving time is March 10, 2023.

[0009] The monoclonal antibody secreted by the hybridoma cell strain and specific to the mycoplasma mycoides subsp. mycoides small colony is also within the protection scope of the application.

[0010] Further, the application also provides the use of the monoclonal antibody in the preparation of a bovine contagious pleuropneumonia detection reagent.

[0011] Further, the application also provides a bovine contagious pleuropneumonia competitive ELISA detection reagent kit, and the reagent kit contains the monoclonal antibody and an antigen coated ELISA plate composed of amino acids 29 to 166 of the extracellular region of a mycoplasma mycoides subsp. mycoides small colony false protein, and the amino acid sequence of the antigen composed of amino acids 29 to 166 of the extracellular region of the mycoplasma mycoides subsp. mycoides small colony false protein is shown as SEQ ID NO. 1.

[0012] Preferably, the reagent kit further contains HRP-labeled goat anti-mouse IgG, blocking solution, washing solution, diluent, color developing solution and termination solution.

[0013] Preferably, the antigen coating concentration is 1 ug / ml, 100 ul per well is coated at 4 DEG C overnight, the serum to be detected is diluted by 20 times; the ascites is used at a concentration of 1:1250, the serum to be detected is mixed with the ascites in equal volume, 100 ul per well is incubated at 37 DEG C for 1 hour; the HRP-labeled goat anti-mouse IgG is used at a concentration of 1:5000, 100 ul per well is incubated at 37 DEG C for 1 hour; the TMB color developing solution is 50 ul per well, color developing is performed at 37 DEG C for 10 minutes in the dark; and 2M sulfuric acid is used as the termination solution, 50 ul per well is terminated.

[0014] Further, the application also provides the use of the reagent kit in the preparation of a bovine contagious pleuropneumonia detection reagent.

[0015] Compared with the prior art, the application has the following beneficial effects:

[0016] For CBPP competition ELISA detection method, the screening of monoclonal antibody is the basis. Foreign researches all use Mmm whole bacteria as immunogen for immunological screening, and this screening method has great randomness, and even if the same scheme is used, it cannot guarantee that the same antibody is obtained. In addition, at present, the foreign CBPP commercial competition ELISA kit uses Mmm whole bacteria lysate as coating antigen, and in China, due to the fact that Mmm can only be operated in a biosafety level 4 laboratory, this method cannot be mass-produced. For the same reason, since Mmm is a class of animal pathogenic microorganisms in China, the method of whole bacteria immunization cannot be used for monoclonal antibody screening. The technical scheme adopted by the present application fully considers the biosafety problem, fully utilizes the bioinformatics technology to analyze the antigen composition, and then obtains the diagnostic marker protein for immunization, and the established competition ELISA detection method is also a protein rather than whole bacteria. Therefore, whether the research and development scheme adopted by the present application, or the antibody screened and the competition ELISA detection method established, are all very different from the existing methods. At the same time, the present application also solves the key technical bottleneck problem, and the specificity of the competition ELISA detection method established by the present application is equivalent to that of the WOAH recommended method (both are more than 99%), and the sensitivity is about 25% higher than that of the WOAH recommended method (the WOAH recommended method is about 63%, and the method of the present application is 88.24%). In addition, since China needs to report the serological prevalence of CBPP to WOAH every year to maintain the CBPP disease-free area state, the establishment of the detection method of the present application is also necessary to maintain the disease-free area state of China. The present application provides an effective technical means for the detection of bovine contagious pleuropneumonia. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 For prokaryotic expression and purification of protein identification;

[0018] Lane M is a protein molecular weight marker, lane 1 is HyP 72-105 protein with MBP tag, lane 2 is HyP 72-105 protein with GST tag, lane 3 is HyP 29-166 protein with MBP tag;

[0019] Figure 2 For cELISA cut-off value determination;

[0020] Figure 3 For cELISA cross-reactivity identification;

[0021] Figure 4 For cELISA detection of Mmm immune sheep serum;

[0022] Figure 5 For cELISA detection method for detection of some areas of China cattle serum;

[0023] In the figure, serum from different regions is represented by different shapes, with bee colony diagrams and violin diagrams used to show the distribution characteristics of the samples. Detailed Implementation

[0024] The present invention will be further illustrated below through experiments and embodiments. It should be understood that these embodiments are for illustrative purposes only and do not limit the scope of protection of the present invention.

[0025] Example 1

[0026] 1. Materials and Methods

[0027] 1.1 Serum

[0028] Known CBPP positive serum, known CBPP negative serum, bovine mycoplasma positive serum, bovine nasal mycoplasma positive serum, agalactiae positive serum, Escherichia coli positive serum, IBRV positive serum, and Mmm immune sheep serum are preserved in our laboratory. BVDV positive serum, bovine tuberculosis positive serum, and bovine paratuberculosis positive serum were kindly donated by the relevant team at the Harbin Veterinary Research Institute. Locally collected serum was sourced from Heilongjiang, Inner Mongolia, Guangxi, and Yunnan provinces.

[0029] 1.2 Screening and Expression of Immunogens

[0030] The complete protein sequences encoding Mmm PG1 were obtained from the NCBI database (GenBank: BX293980.2), totaling 1016 proteins. After transmembrane region analysis, 18 proteins were identified, each containing two or more transmembrane regions, with extracellular regions exceeding 100 amino acids, and subcellularly localized on the cell membrane. Following BLAST analysis, the extracellular region of a pseudoprotein (GenBank: CAE77242.1) was selected as a candidate diagnostic marker. Finally, the extracellular regions containing amino acids 29 to 166 (LIKNTHNNNNNTVLKLYQDGNGSGGSGSSGGSGGAGSNGGSGTSGTSGTGANGGSTTTPGTPAKPEISKEFSTFKDKAEKKIKETLEKVQKRATEIIDQEIAKLKKLSDSTEKSEEYFKNLQKKVYLTELKKHFSNKE, SEQ ID NO.1) and 72 to 105 (SGTSGTGANGGSTTTPGTPAKPEISKEFSTFKDK, SEQ ID NO.2) of the pseudoprotein were selected as the target proteins, and these two proteins were named HyP. 29-166 and HyP 72-105 HyP 29-166The sequence was sent to Jilin Kumei Biotechnology Co., Ltd. for gene synthesis. Then, this sequence was cloned into the pMAL-c5x vector (New England Biolabs, NEB) via EcoR V restriction site restriction to construct the prokaryotic expression plasmid pMAL-HyP. 29-166 HyP 72-105 The sequence was sent to Jilin Kumei Biotechnology Co., Ltd. for gene synthesis. This sequence was cloned into the pMAL-c5x vector via EcoRV restriction site to construct the prokaryotic expression plasmid pMAL-HyP. 72-105 The prokaryotic expression plasmid pGEX-HyP was constructed by cloning into the pGEX-6p-1 vector (General Electric Company, GE) via BamHI and SalI restriction sites. 72-105 The vector construction was performed according to the instructions for the Novizan ClonExpress Ultra One Step Cloning Kit. Expression and purification were carried out as per the product instructions, and this protein was used as the ELISA coating antigen.

[0031] 1.3 Screening and preparation of monoclonal antibodies

[0032] The preparation of monoclonal antibodies is as follows: and Milstein's method was used to ( G., and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-497). Briefly, 5 six-week-old female BALB / c mice (provided by the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were immunized subcutaneously with 50 ug of recombinant protein expressed by pGEX-6p-1 vector mixed with Freund's complete adjuvant at a dose of 50 ug protein per mouse in multiple sites. The mice were boosted every 2-4 weeks after the first immunization with 50 ug of recombinant protein expressed by pGEX-6p-1 vector mixed with Freund's incomplete adjuvant at the same volume, subcutaneously in multiple sites per mouse. Starting from the second immunization, the mice were bled from the tail tip after each immunization, and the serum titers were determined by ELISA plates coated with the same recombinant protein expressed by pMAL-c5X vector as reported in the literature (Xu, Q. Y., Sun, E. C, Yang, T., Feng, Y. F., Geng, H. W., Wei, T., Li, J. P., Sun, L., Sun, J., Xi, N., Zhang, Q., Wang, H. X., Liu, E. Z., Bu, Z. G., and Wu, D. L. (2015) Isolation of a Bluetongue virus group-specific monoclonal antibody and application to a diagnostic competitive ELISA. Appl Microbiol Biotechnol 99, 729-739). Cell fusion was performed 3 days after the mice were boosted, and the feeder cells were prepared from the peritoneal cells of non-immunized mice one day before fusion. The next day, the mice were euthanized, and the spleen cells were fused with SP2 / 0 myeloma cells at a ratio of 5-10: 1 using polyethylene glycol (PEG 4000; Sigma Aldrich). The hybridoma cells were resuspended in HAT medium (containing 20% FBS, 100 mg / mL streptomycin, 100 IU / L penicillin, 100 mM hypoxanthine, 16 mM thymidine, and 400 mM aminopterin) and inoculated into 96-well plates. Six days later, the fusion cells were changed to HT medium (containing 20% FBS, 100 mg / mL streptomycin, 100 IU / L penicillin, 100 mM hypoxanthine, and 16 mM thymidine).After screening by HAT / HT medium, the positive cells were subcloned by limited dilution for 2-3 rounds, and the supernatant of the cells was detected by indirect ELISA using the pMAL-c5X vector expressed protein. When the positive rate of the supernatant in the wells used for screening reached 100%, the screening was completed.

[0033] 1.4 Indirect ELISA method

[0034] The present application uses indirect ELISA for mouse serum and ascites titer determination, and the specific method is referred to Xu et al. and modified appropriately (Xu, Q. Y., Sun, E. C., Yang, T., Feng, Y. F., Geng, H. W., Wei, T., Li, J. P., Sun, L., Sun, J., Xi, N., Zhang, Q., Wang, H. X., Liu, E. Z., Bu, Z. G., and Wu, D. L. (2015) Isolation of a Bluetongue virus group-specific monoclonal antibody and application to a diagnostic competitive ELISA. Appl Microbiol Biotechnol 99, 729-739). Briefly, 5 μg / mL of affinity-purified pMAL-c5X vector-expressed recombinant protein was coated at 100 ul / well at 4°C overnight. The coated plate was incubated with the supernatant of the hybridoma cell culture at 37°C for 1 hour, then incubated with 1:5000 diluted goat anti-mouse IgG (h+L) horseradish peroxidase (HRP) labeled antibody (Sigma) at 37°C for 1 h, followed by color development with o-phenylenediamine substrate solution (Sigma) at 37°C for 10 minutes in the dark. Finally, the reaction was terminated with 2M H2SO4, and the OD450 nm value was read. 450nm absorbance value.

[0035] 1.5 Preparation and titer determination of ascites

[0036] After the selected hybridoma cells were selected, 10-12 week-old BALB / c mice were injected intraperitoneally with Freund's incomplete adjuvant at 0.5 ml per mouse, and one week later, the selected hybridoma cells were injected into the mice at 1×10 5 The selected hybridoma cells were injected into the abdominal cavity of the mice, and after the abdomen of the mice was obviously swollen, the ascites was collected. The ascites supernatant was diluted from 1:1000, and the OD450 nm value was determined. The OD450 nm value ≥ 0.2, P / N value ≥ 2.1, and the highest dilution multiple were the optimal dilution titer. After the ascites titer was determined, it was stored at -80°C for standby.

[0037] 1.6 Identification of Monoclonal Antibody Subtypes

[0038] The subclass identification of the screened monoclonal antibodies was performed using the SBA Clonotyping System-HRP Antibody Subclass Identification Kit (SouthernBiotech). For specific operating procedures, please refer to the product instructions.

[0039] 1.7 Establishment of Competitive ELISA Detection Methods

[0040] The optimal operating conditions were determined by serially diluting the coating antigen, screened monoclonal antibody, serum to be tested, and enzyme-labeled antibody. After determining the optimal reaction conditions, 614 negative serum samples and 51 positive serum samples were tested, and the cut-off value of the detection method was calculated using GraphPad Prism software. The specificity and sensitivity of the detection method were also calculated.

[0041] 1.8 Specificity validation of competing ELISA detection methods

[0042] The established ELISA detection method was used to detect CBPP positive serum, CBPP negative serum, bovine mycoplasma positive serum, bovine nasal mycoplasma positive serum, agalactiae positive serum, Escherichia coli positive serum, IBRV positive serum, BVDV positive serum, bovine tuberculosis positive serum, and bovine paratuberculosis positive serum to determine the cross-reactivity of the detection method with other relevant bovine disease positive serum.

[0043] 1.9 Detection of serum from immunized animals

[0044] The established cELISA detection method was used to detect the serum of 6 Mmm immunized sheep preserved in the laboratory, so as to analyze the seroconversion status of the immunized animals' serum.

[0045] 1.10 Compatibility with commercially available reagent kits

[0046] The IDEXX Mmm serum antibody detection kit and our established detection method were used to simultaneously detect 52 bovine serum samples and 79 immunized sheep serum samples. The Kappa values ​​of the two detection methods were then calculated, and their correlation was compared.

[0047] 1.11 Clinical Sample Testing

[0048] Heilongjiang, Inner Mongolia, Yunnan, and Guangxi are located in my country's border regions and are considered high-risk areas for CBPP (Caucasian Bovine Spongiform Pathogen) transmission. Monitoring bovine serum samples from these regions is crucial for preventing the introduction of exogenous CBPP and maintaining CBPP-free status. Therefore, having successfully established a cELISA detection method, we collected bovine serum from these regions for CBPP serum antibody testing.

[0049] 2Results

[0050] 2.1 Expression of proteins

[0051] According to the amino acid sequence of 72-105 of the extracellular region of the pseudo-protein, a gene was synthesized and inserted into pMAL-c5X vector and pGEX-6p-1 vector, and the recombinant plasmids were named as pMAL-HyP 72-105 and pGEX-HyP 72-105 respectively. In addition, according to the amino acid sequence of 29-166 of the extracellular region of the pseudo-protein, a gene was synthesized and inserted into pMAL-c5X vector, and named as pMAL-HyP 29-166 . The pMAL-HyP 72-105 , pGEX-HyP 72-105 and pMAL-HyP 29-166 plasmids were subjected to prokaryotic expression and purification according to the procedures provided by NEB and GE companies respectively, and the purified three proteins were subjected to SDS-PAGE analysis, which showed that the three proteins were successfully expressed and good purification results were obtained, and finally the HyP 72-105 protein with MBP tag and GST tag and the HyP 29-166 protein with MBP tag were obtained. Figure 1 .

[0052] 2.2 Preparation and identification of monoclonal antibody 3A8

[0053] Mice were immunized with GST-HyP 72-105 as immunogen, and monoclonal antibody screening was performed according to the foregoing method, and finally a hybridoma cell strain stably secreting anti-HyP 72-105 protein was obtained, and was named as Mmm-3A8. The monoclonal antibody secreted by Mmm-3A8 hybridoma cell strain has IgG1 heavy chain and κ chain. The antibody titer in ascites is 10 5 . The Mmm-3A8 hybridoma cell strain is preserved in China General Microbiological Culture Collection Center, and its microbial preservation number is CGMCC NO.45502.

[0054] 2.3 Establishment and verification of cELISA detection method for detecting Mmm serum antibody

[0055] A cELISA detection method was established for detecting CBPP positive bovine serum by using MBP-HyP 29-166 protein as coating antigen and 3A8 monoclonal antibody as detection antibody. The MBP-HyP 29-166The optimal coating concentration of protein, the use concentration of 3A8 antibody, the use concentration of serum to be detected, and the use concentration of goat anti-mouse IgG / HRP are determined by a double array. The optimal ELISA conditions are as follows: the optimal antigen coating concentration is 1 ug / ml, 100 ul of each well is coated at 4°C overnight; the optimal serum dilution multiple is 20 times of dilution; the optimal ascites use concentration is 1:1250, the serum to be detected is mixed with ascites in equal volume, 100 ul of each well is incubated at 37°C for 1 hour; the optimal enzyme-labeled antibody use concentration is 1:5000, 100 ul of each well is incubated at 37°C for 1 hour; the TMB substrate is 50 ul of each well, which is colored at 37°C for 10 minutes in the dark; and 2M sulfuric acid is used as a stop solution, 50 ul of each well is stopped.

[0056] In order to determine the cut off value of the cELISA detection method, 51 known CBPP positive sera and 614 known CBPP negative sera are detected by the established cELISA method. The results are analyzed by GraphPad Prism statistical software, and it is shown that PI%≥60% is determined as positive, and PI%<58 is determined as negative. When the cutoff value is selected, the sensitivity of the cELISA is 88.24% (76.62% to 94.49%) and the specificity is 100% (99.38% to 100.0%) at a confidence level of 95%. Figure 2 )。

[0057] In order to further verify that the cELISA detection method can specifically detect CBPP positive serum and has no cross reaction with other related bovine disease positive serum, the known positive CBPP serum, the known negative CBPP serum, the mycoplasma bovis positive serum, the bovine nasal mycoplasma positive serum, the mycoplasma paralubricum positive serum, the escherichia coli positive serum, the IBRV positive serum, the BVDV positive serum, the bovine tuberculosis bacillus positive serum, and the bovine paratuberculosis positive serum are detected by the established ELISA detection method. The results show that the PI% of the CBPP positive serum is more than 60%, and the PI% of the other disease positive serum and the CBPP negative serum does not exceed 60%. This result shows that the detection method established in the application can specifically detect the CBPP positive infection animal serum, and has no cross reaction with the non-CBPP infection animal serum. Figure 3 )。

[0058] The sera of 6 sheep immunized by the Mmm inactivated vaccine are detected by the cELISA established in the application, and a total of 90 sera are detected. Generally, the serum of the immunized animal is positively converted at 2 weeks to 4 weeks after immunization, and the antibody of the immunized animal is continuously maintained at a high level until 117 days after immunization. Figure 4 )。

[0059] The CBPP serum antibody detection kit produced by IDEXX Company can reflect the level of the established detection method to a certain extent compared with the product. 79 immune sheep sera and 52 bovine sera were used to compare the two detection methods (Table 1). Among them, 55 sera were negative by both detection methods, 72 sera were positive by both detection methods, and 4 sera were negative by the cELISA detection method established by the application and positive by the IDEXX Company kit. According to this, the Kappa value of the two detection methods is 0.94, which indicates that the two detection methods have high correlation.

[0060] Table 1 Correlation analysis of two methods

[0061]

[0062] 2.4 Detection of clinical samples

[0063] A total of 1011 bovine serum samples collected in Heilongjiang (528), Guangxi (176), Yunnan (176) and Inner Mongolia (131) in 2022 were monitored by the established detection method, and all the detected samples were negative. Figure 5 )

Claims

1. A hybridoma cell line secreting monoclonal antibodies specific for Mycoplasma mycoides subsp. mycoides (Mmm), characterized in that, The hybridoma cell strain is named Mmm-3A8, and is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Huayuancun, Beijing, China, and the microbial preservation number is CGMCC NO. 45502, and the preservation time is March 10, 2023.

2. The monoclonal antibody specific to the small colony of Mycoplasma mycoides subsp. mycoides produced by the hybridoma cell strain of claim 1.

3. The use of the monoclonal antibody of claim 2 in the preparation of a reagent for detecting bovine contagious pleuropneumonia.

4. A competitive ELISA test kit for bovine contagious pleuropneumonia, characterized in that, The kit contains the monoclonal antibody of claim 2 and an antigen-coated ELISA plate composed of amino acids 29-166 of the extracellular region of the small colony of Mycoplasma mycoides subsp. mycoides false protein, and the amino acid sequence of the antigen composed of amino acids 29-166 of the extracellular region of the small colony of Mycoplasma mycoides subsp. mycoides false protein is shown as SEQ ID NO.

1.

5. The competitive ELISA test kit for contagious bovine pleuropneumonia according to claim 4, wherein, The kit also includes HRP-labeled goat anti-mouse IgG, blocking solution, washing solution, diluent, color developing solution, and termination solution.

6. The competitive ELISA test kit for contagious bovine pleuropneumonia according to claim 5, wherein, The antigen coating concentration is 1 ug / ml, 100ul per well, 4℃ overnight coating; the serum to be tested is diluted 20 times; the ascites concentration is 1:1250, the serum to be tested is mixed with the ascites in equal volume, 100ul per well, 37℃ incubation for 1 hour; the HRP-labeled goat anti-mouse IgG concentration is 1:5000, 100ul per well, 37℃ incubation for 1 hour; TMB color developing solution is 50ul per well, 37℃ color developing for 10 minutes in the dark; 2M sulfuric acid is the termination solution, 50ul per well.

7. The use of the kit of any one of claims 4-6 in the preparation of a reagent for detecting bovine contagious pleuropneumonia.

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