Hybridoma cell strain secreting anti-salmonella abortus groEL protein monoclonal antibody, monoclonal antibody and application thereof

Abstract

The invention discloses a hybridoma cell strain secreting an anti-salmonella abortus groEL protein monoclonal antibody, the monoclonal antibody and application of the hybridoma cell strain. The hybridoma cell strain is named as E11, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the strain preservation number of the hybridoma cell strain is CGMCC No.46349. According to the present invention, the salmonella universal cELISA antibody detection method capable of being applied to different animals is established by using the E11 MAb secreted by the hybridoma cell strain secreting the anti-horse salmonella abortus groEL protein monoclonal antibody and the horse salmonella abortus groEL protein; the salmonella comprises salmonella abortus (S.Abortuqui), salmonella typhimurium (S.Typhi), salmonella dublin (S.Dublin) and salmonella enteritidis (S.Enteridis), so that the method disclosed by the invention has the advantages of good specificity and broad spectrum. The invention provides an effective technical means for diagnosis, prevention and control of salmonellosis of different animals clinically.

Description

Technical Field

[0001] The present invention relates to a hybridoma cell line secreting monoclonal antibodies against equine Salmonella abortus groEL protein, and also relates to the monoclonal antibodies secreted by the hybridoma cell line and applications thereof. The present invention belongs to the field of medical technology. Background Art

[0002] Salmonellosis is a common foodborne zoonosis worldwide. Its pathogen is Salmonella. This pathogen can infect a variety of animals, including poultry (chickens, ducks, and geese), pigs, cattle, sheep, horses, donkeys, and rabbits. It can also cause a variety of symptoms in humans, including gastroenteritis, food poisoning, typhoid fever, diarrhea, and bacteremia. It is a significant zoonotic pathogen.

[0003] Salmonella spp. include Salmonella enterica ( S. enterica ) and Salmonella bongoreum ( S. bongori ) species, the former is divided into six subspecies, with only the first subspecies infecting warm-blooded animals. It can be further divided into typhoid and paratyphoid. Based on the range of host infection, they can be divided into two major categories: host-adapted serotypes and host-non-adapted serotypes. The former are pathogenic only to the hosts they are adapted to. For example, Salmonella pullorum and Salmonella gallinarum only infect poultry, and Salmonella equi abortus only infects horses, donkeys, and other equine species. The latter are pathogenic to multiple hosts. For example, Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Dublin can infect poultry, cattle, sheep, pigs, horses, donkeys, and other different animals.

[0004] Salmonella gallinarum is currently a major pathogen threatening my country's poultry industry, causing significant economic losses. Therefore, the elimination of Salmonella from poultry farms is crucial. Numerous Salmonella serotypes can cause chicken infections, including Salmonella pullorum, Salmonella gallinarum, Salmonella enteritidis, Salmonella typhimurium, and Salmonella postivirus. Epidemiological studies have shown that flocks on farms can harbor single or mixed infections with multiple Salmonella species. In my country, the predominant serotypes of Salmonella in poultry over the past decade have been Salmonella pullorum, Salmonella enteritidis, and Salmonella typhimurium. Currently, large-scale poultry farms generally eliminate Salmonella by conducting pathogen and antibody testing to eliminate chickens and flocks that test positive. The commonly used serological method, slide agglutination, is inexpensive but has low sensitivity, making complete eradication difficult. ELISA methods are sensitive. Chinese patent application number 201811381001.X discloses a competitive ELISA method based on the IpaJ gene for detecting Salmonella pullorum. However, because a certain proportion of Salmonella pullorum lack the IpaJ gene, this method can miss detection results, resulting in long-term Salmonella pullorum infection in chicken flocks and inability to completely eliminate the disease. Furthermore, chicken flocks can also be infected with other Salmonella species, such as Salmonella Enteritidis and Salmonella Typhimurium. Salmonella elimination in chicken flocks requires the use of ELISA methods for a wider range of Salmonella species, significantly increasing the workload and cost of purification, hindering the successful implementation of Salmonella elimination in chicken flocks.

[0005] Salmonellosis in cattle is primarily caused by infections with Salmonella Typhimurium, Salmonella Enteritidis, and Salmonella Dublin. It is characterized by diarrhea and septicemia, and is mostly sporadic in adult cattle, with a higher incidence in calves. It can also cause miscarriage in pregnant cows. Salmonellosis in cattle can occur year-round, but is more common during rainy and humid seasons. It is often sporadic or endemic, and is more common in adult cattle than in calves. Currently, diagnosis of the disease relies primarily on isolation of the pathogenic bacteria, with relatively little research on ELISA antibody and antigen detection.

[0006] Paratyphoid fever in piglets is a common disease in piglets caused by Salmonella choleraesuis, Salmonella typhimurium, and Salmonella enteritidis. It occurs most frequently in piglets around the time of weaning and has a high incidence and mortality rate. Laboratory diagnostic methods include isolation and identification of the pathogen and quantitative PCR. Isolation and identification of the pathogen is commonly used in veterinary clinical practice, but antigen detection methods such as AC-ELISA and colloidal gold, as well as antibody detection methods such as ELISA, are relatively scarce.

[0007] Equine abortus Salmonellosis has recently broken out in East, North, and Northwest my country, primarily causing mid- to late-term abortions in pregnant horses and donkeys, with abortion rates ranging from 30% to 100%. Therefore, to address this outbreak, there is an urgent need for highly specific, sensitive, and high-throughput detection methods. We have previously developed the iELISA (Application Number CN202010148906.3) and cELISA (Application Number CN202111444112.2) antibody detection methods specific for Salmonella enterica equi. These methods effectively detect antibodies against Salmonella enterica equi, demonstrating their importance in diagnosing Salmonella enterica equi infection. However, the highly specific iELISA and cELISA methods detect only Salmonella enterica equi, but are unable to detect infections in other animals or other Salmonella species.

[0008] The primary route of human infection with Salmonella is through the consumption of contaminated animal products (meat, eggs, and milk). Salmonella infection in animals not only causes losses to the livestock industry but also poses a significant risk to human public health. Therefore, the etiological and serological diagnosis of salmonellosis in animals is crucial. In addition to conventional bacterial isolation and nucleic acid testing, pathogen detection methods include antigen detection methods such as AC-ELISA and colloidal gold. Conventional bacterial isolation methods are time-consuming and labor-intensive, with long cycle times, making them unsuitable for testing large numbers of samples. Among nucleic acid detection methods, fluorescent quantitative PCR offers high sensitivity and is crucial for disease diagnosis. However, it places high demands on the technicians involved and is susceptible to contamination during the testing process. Furthermore, a fluorescent quantitative PCR instrument is required, which is expensive and not widely available, making it inconvenient to use. Compared with bacterial isolation, the AC-ELISA method has higher sensitivity and is easier to achieve high-throughput detection. ELISA does not require high operator requirements and is simpler to operate. The microplate reader used is also cheaper than fluorescent quantitative PCR. In addition, regarding the storage and transportation of the test kit, the generally available ELISA antibody or antigen products are also cheaper than the corresponding nucleic acid detection products. Therefore, the ELISA method has greater advantages than the fluorescent quantitative method. Serological detection methods have higher sensitivity and mainly include iELISA and cELISA. The cELISA method has a great advantage over iELISA. It is not restricted by the animal source of the serum and can therefore be applied to antibody detection in different animal sera. There are many types of Salmonella, which can be divided into more than 2,600 serotypes. Therefore, the establishment of a universal cELISA antibody detection method for Salmonella is extremely important for the diagnosis of salmonellosis in different animals. Summary of the Invention

[0009] The purpose of the present invention is to provide a hybridoma cell line secreting a monoclonal antibody against groEL protein of Salmonella abortus equi, the monoclonal antibody thereof and application thereof.

[0010] In order to achieve the above object, the present invention adopts the following technical means: The present invention generates a hybridoma cell line, E11, by immunizing mice with inactivated Salmonella abortus antigen. Pull-down assays using the E11 monoclonal antibody secreted by this cell line and Salmonella abortus antigens revealed that the E11 monoclonal antibody recognizes the Salmonella abortus groEL protein (heat shock protein). This protein is highly conserved among Salmonella species (99.1-100% homology) and has low homology with other bacteria. Antigenically, this protein is an outer membrane protein with a monomer molecular weight of approximately 60 kDa. Two rings composed of seven subunits stack together to form a tetradecamer. This antigenic component accounts for a significant proportion of the total bacterial composition, making it a dominant antigen and therefore suitable as a target for Salmonella antigen or antibody diagnosis.

[0011] A kind of secretion anti-equine abortion Salmonella of the present invention ( Salmonella Abortusequi, abbreviated as S. The hybridoma cell line is named E11 and classified as a hybridoma cell line (Mus musculus). It is deposited in the General Microbiology Center of the China Culture Collection of Microorganisms, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. Its culture deposit number is CGMCC No. 46349, and the deposit date is April 22, 2025.

[0012] The monoclonal antibody against groEL protein of Salmonella abortus secreted by the hybridoma cell line is also within the protection scope of the present invention.

[0013] Furthermore, the present invention also proposes the use of the hybridoma cell line and the monoclonal antibody in preparing a reagent for detecting Salmonella.

[0014] Among them, preferably, the Salmonella includes Salmonella abortus, Salmonella typhimurium ( Salmonella Typhi, abbreviated as S. Typhi), Salmonella Dublin ( Salmonella Dublin, abbreviated as S. Dublin) and Salmonella Enteritidis ( Salmonella Enteritidis, abbreviated as S. Enteritidis).

[0015] Furthermore, the present invention also provides a cELISA kit for detecting Salmonella, wherein the kit comprises the monoclonal antibody against groEL protein of Salmonella abortus equi.

[0016] Among them, preferably, the kit also includes a groEL protein-coated enzyme-labeled plate, a blocking solution, a PBST washing solution, an HRP-labeled anti-mouse IgG enzyme-labeled secondary antibody, a color development solution and a stop solution, and the amino acid sequence of the groEL protein is shown in SEQ ID NO.1.

[0017] Among them, preferably, the coating concentration of the groEL protein is 1 μg / ml, the dilution of the anti-Salmonella abortus groEL protein monoclonal antibody is 0.2 μg / ml, and the concentration of the HRP-labeled anti-mouse IgG enzyme-labeled secondary antibody is 1:10000.

[0018] Finally, the present invention also proposes the use of the cELISA kit in the preparation of a reagent for detecting Salmonella, wherein preferably, the Salmonella includes Salmonella abortus equi ( S. Abortusequi), Salmonella typhimurium ( S. Typhi), Salmonella Dublin ( S. Dublin), Salmonella Enteritidis ( S. Enteritidis).

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses E11 MAb secreted by a hybridoma cell line secreting monoclonal antibodies against equine abortions groEL protein and equine abortions groEL protein to establish a universal cELISA antibody detection method for Salmonella that can be applied to different animals. The Salmonella include equine abortions ( S. Abortusequi), Salmonella typhimurium ( S. Typhi), Salmonella Dublin ( S. Dublin), Salmonella Enteritidis ( S. Enteritidis ), so the method has good specificity and broad spectrum. The invention provides an effective technical means for the diagnosis and prevention of salmonellosis in different animals in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the SDS-PAGE result of the purified E11 monoclonal antibody; Wherein, M: protein marker; 1-12: monoclonal antibodies in different elution tubes; 13: ascites before purification; 50kDa is the monoclonal antibody heavy chain, 25kDa is the monoclonal antibody light chain; Figure 2 This is the result of antibody titer determination after E11 purification; Figure 3 This is the identification result of the dominant antigen of Salmonella; Among them, M: protein marker; 1: E11 monoclonal antibody; 2: E11 monoclonal antibody + Salmonella antigen; 3: Salmonella antigen; Figure 4 This is the amplification result diagram of the groEL target gene; Wherein, M: nucleic acid marker; 1-3: PCR amplification products; 4: negative control; Figure 5 Figure 1 is the SDS-PAGE analysis result of the soluble expression of recombinant groEL protein; Wherein, M: protein marker; 1: supernatant after recombinant bacteria were broken; 2: precipitate after recombinant bacteria were broken; Figure 6 Figure 1 is the Western blot analysis result of recombinant groEL protein; Wherein, M: protein marker; 1: supernatant after recombinant bacteria were broken; 2: precipitate after recombinant bacteria were broken; Figure 7 This is a diagram showing the validation results of the competitive activity of E11 monoclonal antibody; Figure 8 Optimize OD for different E11 monoclonal antibody concentrations and anti-mouse secondary antibody dilutions 450nm value; Figure 9 This is the N / P value result diagram under different E11 monoclonal antibody concentrations and anti-mouse secondary antibody dilution optimization; Figure 10 This is the specificity validation result of the Salmonella cELISA antibody detection method. DETAILED DESCRIPTION

[0021] The experimental method of the present invention is described in detail below in conjunction with the examples to more clearly illustrate its technical features and implementation steps. It should be understood by those skilled in the art that the embodiments are merely typical examples of the present invention and do not constitute any limitation to the scope of the present invention. Without departing from the spirit and scope of the present invention, the technical details may be adjusted or replaced, and these adjustments and replacements all fall within the scope of protection of the present invention.

[0022] Example 1 Preparation of monoclonal antibodies 1. Materials 1.1 Immunogen: Inactivated Salmonella abortus 180316H.AES.G antigen (CGMCC No. 18341, documented in patent application CN111100817A, entitled "Equine-derived Salmonella abortus strain and its use in the preparation of an inactivated Salmonella abortus vaccine"). Isolated, identified, and provided by the Equine Infectious Diseases and Lentivirus Innovation Team of the Harbin Veterinary Research Institute. Dilution: 1x10 9 CFU / ml.

[0023] 1.2 Myeloma cells SP2 / 0 were provided by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0024] 1.3 Experimental Animals 6- to 8-week-old female Balb / c mice were provided by the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0025] 1.4 Cell culture medium: 1640 medium containing 20% fetal bovine serum and 100 μg / ml penicillin and streptomycin, stored at 2–8°C. 1640 medium was purchased from Sigma; fetal bovine serum was purchased from Ausbian.

[0026] 1.5 Cell freezing solution: Dissolve 10 ml of dimethyl sulfoxide (DMSO) in 90 ml of fetal bovine serum, mix well, and store at 2-8°C until ready for use.

[0027] 1.6 Kit 1.6.1 BCA kit, purchased from Novagen.

[0028] 1.6.2 SBA Clonotyping™ System / HRP, purchased from Southern Biotech.

[0029] 1.7 Other reagents PEG4000, Freund's complete adjuvant, Freund's incomplete adjuvant, and HRP-labeled goat anti-mouse IgG were purchased from Sigma; TMB colorimetric solution was purchased from Thermo Fisher Scientific.

[0030] 2 Methods 2.1 Construction of hybridoma cells 2.1.1 Animal immunization 2.1.1.1 Primary Immunization: Inactivated Salmonella abortus antigen was emulsified with an equal volume of Freund's complete adjuvant as the immunogen and injected subcutaneously into the back of 4-6 week old Balb / c mice at a volume of 200 μl per mouse.

[0031] 2.1.1.2 Secondary Immunization Three weeks after the first immunization, the inactivated antigen was mixed with an equal volume of Freund's incomplete adjuvant and emulsified as the immunogen. The second immunization was performed by intraperitoneal injection according to the immunization method and dosage of the first immunization.

[0032] 2.1.1.3 Three weeks after the second immunization, the inactivated Salmonella antigen was mixed with an equal volume of Freund's incomplete adjuvant and emulsified as the immunogen. The third immunization was carried out according to the immunization method and dosage of the second immunization.

[0033] 2.1.1.4 Three days before the fourth immune cell fusion, inoculate mice with inactivated Salmonella antigen as the immunogen via intraperitoneal injection at 200 μl per mouse.

[0034] 2.1.2 Myeloma Cell Preparation: 1-2 days before fusion, expand the myeloma cells to a well-developed logarithmic growth phase. On the day of fusion, discard the culture medium and gently rinse twice with serum-free 1640 medium. Gently remove the cells from the flask wall with 15 ml of 1640 basal medium. Take a small amount of the osteoblast cell suspension, count it using a cell counter, and prepare for fusion.

[0035] 2.1.3 Preparation of immune splenocytes 2.1.3.1 Before fusion, blood was collected from the eyeballs of the boosted immunized mice and sacrificed to prepare positive serum. The serum was soaked in 75% alcohol for 5 minutes and then placed on a clean bench.

[0036] 2.1.3.2 Secure the mouse in a mouse rack, open the abdominal cavity aseptically, separate the connective tissue, and remove the spleen. Place the spleen in a dish containing 15 ml of 1640 basal culture medium. Use a sterile syringe to aspirate the culture medium and gently blow out the spleen cells. Repeat this operation 3 to 4 times.

[0037] 2.1.3.3 Transfer the spleen cell suspension to a 50ml centrifuge tube, add 1640 basal culture medium to approximately 30ml, and mix thoroughly. Count the cell suspension using a cell counting plate and set aside.

[0038] 2.1.4 Fusion of splenocytes and myeloma cells 2.1.4.1 Preheat HAT medium, 1640 medium, and 1 ml of PEG4000 in a water bath. Prepare 500 ml of sterile water preheated to 42°C.

[0039] 2.1.4.2 Add the logarithmically growing SP2 / 0 cells prepared above to the spleen cells of the immunized mouse at a ratio of 1:8 into a 50ml centrifuge tube and gently invert to mix. Centrifuge at 800 rpm for 10 minutes. Aspirate the supernatant to avoid affecting the fusion efficiency. Gently tap the bottom of the centrifuge tube to ensure that the cells are evenly distributed across the bottom of the tube.

[0040] 2.1.4.3 The fusion process is carried out in a fusion cup filled with 42°C water. 1 ml of PEG 4000 solution preheated at 37°C is added dropwise to a 50 ml centrifuge tube while slowly rotating the centrifuge tube. The addition should be completed within 90 seconds and the tube should be allowed to stand at 37°C for 1-2 minutes.

[0041] 2.1.4.4 Terminate the reaction by slowly and then rapidly adding 1640 basal medium: add 1 ml of 1640 dropwise at the first minute, 1 ml at the second minute, 3 ml at the third minute, 10 ml at the fourth minute, and 10 ml at the fifth minute. Let stand for 2 minutes, gently invert twice, let stand for 7 minutes, centrifuge at 800 rpm for 10 minutes, and discard the supernatant. Gently resuspend the cells in 110 ml of HAT medium and plate 200 μl / well in a 96-well plate containing feeder cells. Incubate in a 37°C, 5% CO2 incubator.

[0042] 2.1.4.5 Change the medium by half after 3 days and by half after 6 days. When the cells have grown to 1 / 4 to 1 / 3 of the bottom area of the well, remove the supernatant for screening and detection and replace with HT culture medium.

[0043] 2.1.5 Screening and cloning of positive hybridoma cell lines: Hybridoma cell culture supernatants were tested using the indirect ELISA method, and positive clones were screened by 2-3 consecutive limiting dilution cloning methods. The cell lines were then expanded and cryopreserved. The indirect ELISA method is as follows: 2.1.5.1 Coating: Dilute the inactivated antigen to 1µg / ml using carbonate buffer (0.05mol / L, pH 9.6) and add 100µl / well to a 96-well enzyme-linked reaction plate. Incubate at 2-8°C overnight.

[0044] 2.1.5.2 Washing: Discard the liquid in the wells and wash the plate three times with PBST (0.01 mol / L, pH 7.4), 250 µl / well. After each wash, invert the plate onto dry filter paper to pat dry the liquid.

[0045] 2.1.5.3 Blocking: Add PBS (0.01 mol / L, pH 7.4) containing 5% skim milk, 200 µl / well, and incubate at 37°C for 2 hours.

[0046] 2.1.5.4 The washing method is the same as that in 2.1.5.2.

[0047] 2.1.5.5 Add the sample to be tested, 100µl / well, and incubate at 37℃ for 1 hour.

[0048] 2.1.5.6 The washing method is the same as that in 2.1.5.2.

[0049] 2.1.5.7 Add secondary antibody: Add 1:10,000 diluted HRP-labeled goat anti-mouse IgG, 100 µl / well, and incubate at 37°C for 30 min.

[0050] 2.1.5.8 The washing method is the same as that in 2.1.5.2.

[0051] 2.1.5.9 Color Development: Add TMB color development solution (100µl / well) and incubate at room temperature (15-25°C) for 5 minutes in the dark.

[0052] 2.1.5.10 Stop by adding 50µl / well of stop solution, gently vortex to mix, and read the OD450nm value at a wavelength of 450nm using a microplate reader (the reading should be completed within 5 minutes after adding the stop solution) and record the result.

[0053] 2.1.5.11 Judgment The test is established when the OD450nm value of the sample to be tested is greater than 0.5 and the P / N value (P / N = OD 450nm The titer was determined to be positive if the value (OD value / negative control OD450nm value) was greater than 2.1, and the corresponding maximum dilution was the titer.

[0054] 2.1.6 Subcloning of Positive Hybridoma Cell Lines Subcloning of positive hybridoma cells was performed using the limiting dilution method, with cloning performed three times until the antibody positivity rate in the cloning wells reached 100%. The obtained positive hybridoma cells were expanded and cultured and then frozen in liquid nitrogen.

[0055] 2.2 Preparation and purification of monoclonal antibodies 2.2.1 Preparation of Ascites Balb / c mice aged 6 to 8 weeks were injected intraperitoneally with 0.5 ml of Freund's incomplete adjuvant. 7 to 10 days after inoculation, 1 to 2.5 × 10 6 10000 rpm for 10 minutes at 4°C. Collect the supernatant.

[0056] 2.2.2 Purification of monoclonal antibodies 2.2.2.1 Sample preparation: Dissolve the prepared ascites at room temperature, mix with 4-5 volumes of binding / washing buffer (20 mmol / L sodium phosphate, pH 7.0), and filter through a 0.45 µm filter.

[0057] 2.2.2.2 Column Packing and Equilibration Load HiTrap protein G filler (approximately 2 ml column bed volume) into a suitable chromatography column and equilibrate with 10 column volumes of binding / wash buffer (20 mmol / L sodium phosphate, pH 7.0) at a flow rate of 1 ml / min.

[0058] 2.2.2.3 Loading: Add the sample to the equilibrated chromatography column at a flow rate of 0.2-1 ml / min, collect the effluent, and repeatedly pass the sample through the column 3-5 times.

[0059] 2.2.2.4 Washing: Use 10-15 column volumes of binding / wash buffer (20 mmol / L sodium phosphate, pH 7.0) to remove non-specifically adsorbed proteins.

[0060] 2.2.2.5 Elution: Perform elution 5-6 times with elution buffer (0.1 mol / L glycine, pH 2.7), with a volume of 1 ml per elution. Collect the flow-through. Immediately after elution, neutralize with neutralization buffer (1 mol / L Tris-HCl, pH 9.0). Add approximately 120 μl of neutralization buffer (1 mol / L Tris-HCl, pH 9.0) per 1 ml of elution flow-through.

[0061] 2.2.2.6 SDS-PAGE Analysis: Mix the purified monoclonal antibodies in different elution tubes with 1 / 4 volume of 5× SDS loading buffer, incubate in a 100°C metal bath for 10 minutes, and perform SDS-PAGE analysis. Take 10 µL of the treated sample and a protein molecular weight marker and perform a 12% SDS-PAGE electrophoresis. Analyze the gel image using BandScan 5.0 software to determine the purity of the monoclonal antibody.

[0062] 2.3 Identification of monoclonal antibodies 2.3.1 Identification of Monoclonal Antibody Subclasses The subclasses of the screened monoclonal antibodies were identified using the SBA Clonotyping™ System / HRP kit according to the manufacturer's instructions.

[0063] 2.3.2 Determination of protein concentration The protein concentration of the purified monoclonal antibody was determined according to the detection method in the BCA kit instructions.

[0064] 2.3.3 Titer Determination: Antibodies purified from hybridoma cell lines (1 mg / mL) were diluted two-fold starting from 1:200 in PBS (0.01 mol / L, pH 7.4) and titered using indirect ELISA.

[0065] 3 Results 3.1 Screening of monoclonal antibody cell lines After three rounds of purification, two hybridoma cell lines that can recognize equi abortion Salmonella antigens were screened.

[0066] 3.2 Identification of monoclonal antibodies 3.2.1 Monoclonal Antibody Subclass Identification The subclasses of the screened monoclonal antibodies were identified according to the SBA Clonotyping™ System / HRP kit instructions. The results (Table 1) showed that the E11 monoclonal antibody subclass was IgG2a, and the 2D2 monoclonal antibody subclass was IgM. To facilitate subsequent antibody purification, only the IgG subclass of E11 was preserved and studied. The hybridoma cell line E11, which secretes anti-S. equi abortus monoclonal antibodies, was deposited with the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. Its culture collection number is CGMCC No. 46349, and the deposit date is April 22, 2025.

[0067] ; 3.2.2 Antibody titer determination Antibody purification was performed according to the HiTrap Protein G HP (GE) instructions. SDS-PAGE results showed that two specific bands appeared after SDS-PAGE electrophoresis, with the heavy chain at approximately 50 kDa and the light chain at approximately 25 kDa. Gel images were analyzed using BandScan 5.0 software. The purity of the purified monoclonal antibody (lanes 3-10) was greater than 98%, indicating that relatively pure antibodies were obtained. For details, see Figure 1 After the purified antibodies were mixed (lanes 3-10) and diluted to 1 mg / ml, the titer of the purified antibodies from the hybridoma cell line E11 was determined by indirect ELISA. The results showed that the titer of the E11 monoclonal antibody was 6.4x10 6 ( Figure 2 ).

[0068] Example 2 Identification and expression of antigens recognized by E11 monoclonal antibody and verification of competitive activity of E11 monoclonal antibody 1 Materials and Methods 1.1 Strains Salmonella abortus strain 180316H.AES.G (CGMCC No. 18341) 1.2 Samples and Reagents Agarose gel DNA recovery kit (Gel Extraction Kit), high-purity plasmid mini kit (Pureplasmid Mini Kit) and others were purchased from Kangwei Century Co., Ltd.

[0069] 1.3 Antigen identification To prepare the S. equi abortus antigen complex, 8 mL of fresh logarithmic-phase S. equi abortus culture was centrifuged at 10,000 rpm for 2 min in a biosafety cabinet. The cells were then harvested and resuspended in 2 mL of sterile PBS, centrifuged, and the supernatant discarded. The cells were then thoroughly washed twice. The cells were then resuspended in 1.5–2 mL of sterile PBS and disrupted by sonication at 39% power for 3 s, 5 s rest, and 5 min. Finally, the bacterial lysate was centrifuged at 10,000 rpm for 2 min at 4°C. The supernatant was collected and the protein concentration was determined using BCA assay and adjusted to 1 mg / mL for later use. Pull-down assays were then performed between the E11 monoclonal antibody and the S. equi abortus antigens. The results were analyzed by mass spectrometry.

[0070] 1.4 Amplification and sequence analysis of target genes Based on the groEL sequence (GenBank: AB033231), a pair of primers were designed: ; The gorEL gene was amplified by PCR using Salmonella abortus (180316H.AES.G) as a template and cloned into the pET28a vector to generate the recombinant vector pET28a-gorEL. The amplified gorEL was sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing. Following sequencing, the amino acid sequence of the S. abortus groEL sequence was compared with that of Salmonella pullorum, Salmonella Enteritidis, Salmonella Galli, Salmonella Dublin, Salmonella Typhimurium, Salmonella Choleraesuis, and Salmonella Paratyphi A, as well as groELs from other bacteria (including Escherichia coli, Streptococcus, Helicobacter pylori, Staphylococcus aureus, Brucella, Bacillus, Pseudomonas, Riemerella anatipestifer, and Pasteurella multocida).

[0071] 1.5 Induced expression, purification and identification of target protein Transform the recombinant plasmid pET28a-gorEL into BL21 and culture at 37℃ for 12-14 hours. Inoculate the positive colonies into 5mL of fresh LB liquid medium containing 1µg / mL Kanamycin resistance and culture at 37℃ with shaking at 170r / min for 16h. Inoculate the colonies into Kan / LB liquid medium at a ratio of 1:100 and culture at 37℃ with shaking at 170r / min until the OD value of the bacterial solution reaches 0. 600nmWhen the pH value was approximately 0.6-0.8, IPTG was added to a final concentration of 0.6 mmol / L at 25°C for induction. After 8 hours of induction, 4 ml of bacterial culture was collected for enrichment and ultrasonically disrupted in 1.5 ml of PBS. After disruption, the pellet was centrifuged at 12,000 rpm at 4°C for 5 minutes. The pellet was rinsed twice with an equal volume of PBS and resuspended in an equal volume of PBS. 40 µL of the supernatant and resuspended pellet were each added with 10 µL of 5x SDS-PAGE Loading Buffer, mixed thoroughly, and boiled at 98°C for 10 minutes. A 20 µL sample was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. Western blot analysis was performed using E1 monoclonal antibody as the primary antibody (1:5000 dilution) and fluorescently labeled anti-mouse IgG as the secondary antibody (1:5000 dilution).

[0072] 1.6 Verification of the competitive activity of E11 mAb (1) The purified groEL protein was coated at 1 μg / ml (100 μl / well) at 4°C for 16 h. (2) After blocking, add Salmonella positive and negative serum (100 μL / well) and incubate at 37°C for 1 h; (3) After washing twice with PBST, add E11 hybridoma cell supernatant and incubate at 37°C for 30 minutes; (4) After washing twice with PBST, add HRP-labeled anti-mouse IgG enzyme-labeled secondary antibody diluted 1:20,000 and incubate at 37°C for 30 minutes; (5) After washing twice with PBST, add substrate (100 μL / well) and develop color at 37°C for 10 minutes; (6) Add 2M H2SO4 to terminate the reaction. 450nm Read the value.

[0073] 2. Results 2.1 Pull-down search for Salmonella antigens Through the pull-down test, compared with the E11 monoclonal antibody group (lane 1), the experimental group (lane 2) showed a specific band of about 60KD ( Figure 3 The specific pull-down band was sent to Beijing BGI Protein Research and Development Center Co., Ltd. for mass spectrometry identification, which confirmed the band to be the groEL protein. Pull-down analysis confirmed that the antigen recognized by the E11 monoclonal antibody was the groEL protein. Comparison of the amino acid sequence of groEL proteins from other Salmonella species and other bacteria revealed that this protein is highly conserved within the Salmonella genus, with 99.1-100% homology, and significantly different from other species. This paves the way for the development of a universal diagnostic kit for salmonellosis.

[0074] 2.2 Cloning of the Salmonella groEL gene The groEL gene was amplified by PCR using the designed primers. The results showed that a band of approximately 1647 bp was amplified ( Figure 4 ), digested with enzymes, ligated into pET28a, and transformed into BL21. A positive plasmid identified by PCR was sequenced, and the sequence obtained confirmed the Salmonella groEL sequence. The positive plasmid was named pET28a-groEL.

[0075] 2.3 Expression and identification of groEL protein SDS-PAGE analysis showed that a protein band with a relative molecular mass of about 60 KDa appeared in the supernatant of the recombinant pET28a-groEL bacteria after disruption, proving that the recombinant protein was expressed and existed mainly in a soluble form ( Figure 5 Western blot analysis showed that the recombinant protein reacted specifically with E11 monoclonal antibody ( Figure 6 ), which is consistent with the SDS-PAGE results, confirming that the recombinant protein has good reactogenicity. The amino acid sequence of the groEL protein is shown in SEQ ID NO.1.

[0076] 2.4 Verification of the competitive activity of E11 mAb The competitive activity of E11 was determined by ELISA. The results showed that when the serum contained Salmonella antibodies, it could effectively block the binding of E11 monoclonal antibody to the coated groEL antigen, thus proving that E11 monoclonal antibody has good competitive activity ( Figure 7 ), which can be used for the subsequent research on the establishment of Salmonella competitive ELISA method.

[0077] Example 3 Establishment and Application of Salmonella cELISA Antibody Detection Method 1. Screening of optimal antibody dilution and enzyme-labeled antibody working concentration The purified groEL protein was coated at 1 μg / ml (100 μl / well) at 4°C for 16 h. After blocking, Salmonella positive and negative serum (100 μL / well) were added and incubated at 37°C for 1 h. After washing twice with PBST, different concentrations of E11 monoclonal antibody were added and incubated at 37°C for 30 min. After washing twice with PBST, HRP-labeled anti-mouse IgG enzyme-labeled secondary antibody of different dilutions were added and incubated at 37°C for 30 min. After washing twice with PBST, substrate (100 μl / well) was added and color was developed at 37°C for 10 min. 2 M H2SO4 was added for termination. 50nm The antibody dilution and enzyme-labeled antibody working concentration were optimized and screened to determine the optimal reaction conditions. The results are as follows: Figure 8As shown. OD values were optimized according to different monoclonal antibody concentrations and anti-mouse secondary antibody dilutions. 450nm Value and N / P value, determine the best conditions, the results are as follows Figure 9 The final conditions were as follows: coating antigen concentration of 1 μg / ml, antibody dilution of 0.2 μg / ml, and anti-mouse secondary antibody concentration of 1:10,000.

[0078] 2. Determination of the critical value of the Salmonella cELISA antibody detection method Thirty Salmonella-negative sera were tested using the optimized cELISA antibody detection method. The inhibition rate (PI) was calculated using the following formula (1-S / N). Based on the PI values of the 30 Salmonella-negative sera, the cutoff value was 45% (mean + 3SD). A PI value greater than 45% was considered positive for Salmonella antibodies, while a value less than 45% was considered negative.

[0079] 3. Specificity test The optimized reaction conditions were used to detect equine infectious anemia virus (EIAV), equine arteritis virus (EAV), equine herpes virus (EHV), equine influenza virus (EIV), Theileria equi ( T.equi ), Babesia equi ( B. caballi ), Streptococcus equine ( S.equi ), Salmonella abortus equi ( S. Abortusequi), Salmonella typhimurium ( S. Typhi), Salmonella Dublin ( S. Dublin), Salmonella Enteritidis ( S. Enteritidis) and other positive sera were tested to evaluate the specificity of the Salmonella cELISA antibody detection method. The results are as follows Figure 10 The results showed that all Salmonella-positive sera were positive, while the test results of other pathogen-positive sera were negative, thus proving that the method has good specificity and broad spectrum.

[0080] 4. Comparison of detection of Salmonella cELISA and equine Salmonella abortus specific ELISA antibodies The Salmonella cELISA of the present invention was used to test 20 Salmonella-positive sera and 20 Salmonella-negative sera, and compared with the equine Salmonella abortus iELISA (patent application number CN202010148906.3) and cELISA (patent application number CN202111444112.2). The results showed that the Salmonella cELISA of the present invention achieved an overall concordance rate of 100% with the other two equine Salmonella abortus iELISA / cELISA methods (Table 2), demonstrating excellent concordance.

[0081] ; Note: “+” represents a positive result, “-” represents a negative result 5. Application of Salmonella cELISA in the detection of Salmonella antibodies in equines The Salmonella cELISA antibody detection method established in the present invention was used to detect 7 farm serum samples, and the results were compared with those of the equine abortion Salmonella iELISA (CN202010148906.3) and cELISA (CN202111444112.2) methods. The results are shown in Table 3. For Farm No. 1, where the equine abortion Salmonella iELISA (CN202010148906.3) and cELISA (CN202111444112.2) methods tested 100% positive, the Salmonella cELISA positive detection rate established by the present invention was also 100%, which also proved that the cELISA of the present invention has good sensitivity and will not cause false negative results; for Farm No. 2 (a Salmonella-negative farm), where the equine abortion Salmonella iELISA (CN202010148906.3) and cELISA (CN202111444112.2) methods tested negative, the cELISA test results established by the present invention were also all negative, which proved that this method has good specificity and will not cause false positive results. For Farms 3-4 (Salmonella equine abortions infection farms) where the Salmonella equine abortions cELISA (CN202111444112.2) method tested negative and the Salmonella equine abortions iELISA (CN202010148906.3) detection rates were 20% and 25%, respectively, the positive detection rates of Salmonella cELISA (CN202111444112.2) were 30% and 75%, respectively, and the sensitivity was also higher than that of the Salmonella equine abortions iELISA (CN202010148906.3). Since the equine Salmonella abortions iELISA (CN202010148906.3) and cELSIA (CN202111444112.2) are only specific for the detection of equine Salmonella abortions antibodies, the Salmonella cELISA (CN202111444112.2) established in the present invention can detect antibodies to different Salmonella species. In addition, the early detection of clinical samples using the Salmonella typhimurium-specific iELISA antibody detection method established by us also confirmed the presence of equine Salmonella abortions and Salmonella typhimurium in some farms. Therefore, for farms 5-7 where both iELISA (CN202010148906.3) and cELISA (CN202111444112.2) tests for Salmonella abortus were negative, the positive result of cELISA (CN202111444112.2) for Salmonella antibodies established in this study was 50%-62.5%, which further proved that this method has higher sensitivity and better spectrum in detecting antibodies to different Salmonella species, and is suitable as a universal diagnosis of Salmonella.

[0082] .

Claims

1. Secretion of anti-equine Salmonella abortus ( Salmonella A hybridoma cell line producing a monoclonal antibody against groEL protein, characterized in that: The hybridoma cell line was named E11 and deposited at the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. Its culture deposit number is CGMCC No. 46349, and the deposit date is April 22, 2025.

2. A monoclonal antibody against groEL protein of Salmonella abortus equi, characterized in that: The monoclonal antibody is secreted by the hybridoma cell line according to claim 1.

3. Use of the hybridoma cell line according to claim 1 in preparing a reagent for detecting Salmonella.

4. Use of the monoclonal antibody according to claim 2 in preparing a reagent for detecting Salmonella.

5. The use according to claim 3 or 4, characterized in that The Salmonella include equine abortion Salmonella, Salmonella typhimurium ( Salmonella Typhi), Salmonella Dublin ( Salmonella Dublin) and Salmonella Enteritidis ( Salmonella Enteritidis).

6. A cELISA kit for detecting Salmonella, characterized in that: The kit comprises the monoclonal antibody against equine abortions groEL protein according to claim 2.

7. The cELISA kit according to claim 6, wherein The kit also includes a groEL protein-coated enzyme-labeled plate, a blocking solution, a PBST washing solution, an HRP-labeled anti-mouse IgG enzyme-labeled secondary antibody, a color development solution, and a stop solution. The amino acid sequence of the groEL protein is shown in SEQ ID NO.

1.

8. The cELISA kit according to claim 6 or 7, wherein The coating concentration of the groEL protein is 1 μg / ml, the dilution of the monoclonal antibody against the equine Salmonella abortus groEL protein is 0.2 μg / ml, and the concentration of the HRP-labeled anti-mouse IgG enzyme-labeled secondary antibody is 1:10,000.

9. Use of the cELISA kit according to any one of claims 6 to 8 in preparing a reagent for detecting Salmonella.

10. The use according to claim 9, characterized in that The salmonella include equi abortion salmonella, typhimurium salmonella, dublin salmonella and enteritidis salmonella.

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