Anti-Salmonella typhimurium antibody, immunomagnetic beads labeled therewith, and applications thereof

Abstract

The present invention discloses an anti-Salmonella typhimurium antibody, immunomagnetic beads labeled therewith, and applications thereof. The present invention provides an anti-Salmonella typhimurium monoclonal antibody comprising a light chain variable region and a heavy chain variable region, wherein LCDR1 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:1, LCDR2 comprises the amino acid sequence LAS, and LCDR3 comprises the sequence shown in SEQ ID NO:2; and HCDR1 to HCDR3 of the heavy chain variable region comprise the sequences shown in SEQ ID NO:3, 4, and 5, respectively. The present invention establishes a Salmonella typhimurium immunomagnetic bead enrichment and separation method based on the specific monoclonal antibody against Salmonella typhimurium. The method is rapid and simple, has a high capture rate, strong specificity, and good sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of pathogen separation, and particularly relates to an anti-Salmonella typhimurium antibody, immunomagnetic beads labeled with the antibody, and applications thereof. Background Art

[0002] Salmonella Typhimurium is a Gram-negative, facultative anaerobic foodborne pathogen that is the leading cause of foodborne bacterial infections worldwide. Salmonella food poisoning, primarily caused by meat, animal offal, milk, and eggs, poses a serious threat to food safety and human health. Ingestion of Salmonella Typhimurium can directly cause systemic illness such as vomiting, gastroenteritis, and severe diarrhea. Without effective treatment, it can lead to intestinal obstruction, gastrointestinal bleeding, intestinal perforation, septic shock, and death.

[0003] Direct detection of Salmonella Typhimurium in food samples can be affected by a variety of opportunistic bacteria and other components. Sample pretreatment is essential for detecting Salmonella Typhimurium in food samples to remove interfering substances and significant background interference from the food, thereby improving the sensitivity and specificity of subsequent detection methods. Furthermore, prior to detection, the target bacteria in the food sample must be concentrated and enriched to reach the detection limit of the detection method. Traditional culture methods require 6-12 hours to reach a concentration that meets the detection limit of subsequent detection methods. Bacterial enrichment techniques, however, can rapidly concentrate the target bacteria in food samples within tens of minutes. Among the many enrichment techniques used in sample processing, immunomagnetic separation based on antigen-antibody reactions is currently a hot topic. This technique offers numerous advantages, such as shortening the incubation time for microbial detection, removing matrix-interfering substances, and reducing the required sample volume. This allows for rapid enrichment of microorganisms. Especially for samples with low initial concentrations, enrichment techniques can improve analytical accuracy, expand the application range of detection technologies, and improve sampling techniques for detecting low-level pathogens or sporadic contamination. This technology has been widely used in the rapid enrichment and detection of pathogenic microorganisms.

[0004] Monoclonal antibodies can specifically recognize and bind to their corresponding antigens. In the immunomagnetic bead separation technology based on antigen-antibody reaction, obtaining specific monoclonal antibodies is the most critical technology. Monoclonal antibodies with high affinity and strong specificity are necessary conditions for immunomagnetic bead technology to achieve high capture rate and high specificity. Monoclonal antibodies are coupled with magnetic beads to prepare immunomagnetic beads. By further optimizing multiple reaction conditions and under the action of a magnetic field, pathogenic microorganisms in the sample can be rapidly, efficiently and specifically enriched, and then used for the separation and detection of target pathogens. Summary of the Invention

[0005] In order to solve the defects of the prior art in directly detecting Salmonella typhimurium in food, which cannot achieve both rapidity, simplicity, specificity and sensitivity, the present invention provides an anti-Salmonella typhimurium antibody, immunomagnetic beads labeled therewith and their application.

[0006] In a first aspect, the present invention provides an anti-Salmonella typhimurium monoclonal antibody comprising a light chain variable region and a heavy chain variable region, wherein LCDR1 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, LCDR2 comprises the amino acid sequence LAS, and LCDR3 comprises the sequence shown in SEQ ID NO: 2; and HCDR1 to HCDR3 of the heavy chain variable region comprise the sequences shown in SEQ ID NO: 3, 4 and 5, respectively.

[0007] In some embodiments, the light chain variable region comprises the sequence shown in SEQ ID NO:6, and the heavy chain variable region comprises the sequence shown in SEQ ID NO:8.

[0008] In some embodiments, the light chain of the monoclonal antibody comprises the sequence shown in SEQ ID NO:7, and the heavy chain comprises the sequence shown in SEQ ID NO:9.

[0009] The second aspect of the present invention provides a polynucleotide encoding the monoclonal antibody of the present invention; preferably, it comprises the light chain nucleotides as described in SEQ ID NO: 10 and / or the heavy chain nucleotides as described in SEQ ID NO: 11.

[0010] The third aspect of the present invention provides a recombinant expression vector comprising the polynucleotide according to the present invention.

[0011] The fourth aspect of the present invention provides a transformant, which comprises the polynucleotide according to the present invention or the recombinant expression vector according to the present invention; preferably, the host thereof is Escherichia coli.

[0012] A fifth aspect of the present invention provides antibody-labeled immunomagnetic beads, wherein the antibody is the monoclonal antibody described in the present invention.

[0013] The immunomagnetic beads can be prepared by conventional methods in the art. In some embodiments, the immunomagnetic beads are activated during preparation. For example, the preparation method includes labeling the monoclonal antibody with activated immunomagnetic beads.

[0014] The immunomagnetic beads can be conventional in the art. In some embodiments, the diameter of the activated immunomagnetic beads is 180 to 2000 nm, preferably 180 nm. For example, Orun Company offers four different sizes of magnetic beads: PM3-020 (180 nm diameter), PM3-050 (750 nm diameter), SM3-P100 (1150 nm diameter), and 70102-5 (2000 nm diameter).

[0015] In some embodiments, the activation method preferably comprises the following steps:

[0016] (1) mixing the immunomagnetic beads with a MEST solution; the pH of the MEST solution is preferably 6;

[0017] (2) Add a mixed solution of EDC and NHS to obtain activated immunomagnetic beads.

[0018] In some embodiments, the volume ratio of the EDC and the NHS in step (2) is 1:1, and the concentration of both is 5 mg / mL.

[0019] A sixth aspect of the present invention provides a method for enriching and isolating Salmonella typhimurium, which comprises using the immunomagnetic beads described in the present invention to enrich and isolate Salmonella typhimurium.

[0020] In some embodiments, the enrichment comprises the following steps:

[0021] (1) Incubating the immunomagnetic beads of the present invention and the sample to be tested in a coupling buffer for 45 to 75 minutes; wherein the coupling buffer is a MEST solution, and the pH of the MEST solution is preferably 7;

[0022] Optionally, the method further includes (2) calculating the capture rate; wherein the capture rate is calculated as follows: capture rate (%) = number of bacteria captured by immunomagnetic beads / (number of bacteria in the supernatant + number of bacteria captured by immunomagnetic beads) × 100%.

[0023] In some embodiments, the volume of the enriched reaction system and the relative usage amount of the immunomagnetic beads are 1 mL:0.1-0.5 mg, preferably 1 mL:0.3-0.5 mg, and more preferably 1 mL:0.4 mg.

[0024] In a seventh aspect, the present invention provides a use of the monoclonal antibodies and immunomagnetic beads of the present invention in enriching and isolating Salmonella typhimurium or preparing a reagent for enriching and isolating Salmonella typhimurium.

[0025] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0026] The reagents and raw materials used in the present invention are commercially available.

[0027] The positive progress effect of the present invention is:

[0028] In order to establish a rapid enrichment and detection method for Salmonella typhimurium in food samples, this study used Salmonella typhimurium to immunize mice, and through cell fusion screening, prepared anti-Salmonella typhimurium monoclonal antibodies with strong specificity and good sensitivity. The anti-Salmonella typhimurium monoclonal antibodies prepared by the present invention have high titers (for example, up to 1:20,480,000), and the monoclonal antibodies have strong specificity; and are suitable for the preparation of monoclonal antibody-based immunomagnetic beads. The prepared immunomagnetic beads have a high capture rate for Salmonella typhimurium, as high as more than 95%; the prepared immunomagnetic beads have strong specificity and no cross-reaction with other common foodborne bacteria; the prepared immunomagnetic beads have good sensitivity, and when the concentration of Salmonella typhimurium in the capture system is as low as 10 2 The immunomagnetic beads of the present invention have good practical application value. When the content of Salmonella typhimurium in food samples, such as milk, is as low as 10 CFU / mL, the capture rate of the immunomagnetic beads is still above 90%. 2 CFU / mL, the capture rate of immunomagnetic beads was still over 85%. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Determination of the optimal particle size for Salmonella typhimurium immunomagnetic beads.

[0030] Figure 2 Determination of the optimal dosage of magnetic beads for Salmonella typhimurium immunoprecipitation.

[0031] Figure 3 Determination of the optimal reaction time for Salmonella typhimurium immunomagnetic beads.

[0032] Figure 4 Evaluation of the specificity of immunomagnetic beads for Salmonella typhimurium.

[0033] Figure 5 Evaluation of the sensitivity of immunomagnetic beads for Salmonella typhimurium.

[0034] Figure 6 To evaluate the effectiveness of capturing Salmonella typhimurium in real samples. DETAILED DESCRIPTION

[0035] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0036] The following examples involve: three types of Salmonella typhimurium (Salmonella typhimurium ATCC14028, Salmonella typhimurium ATCC21484, Salmonella typhimurium ATCC10420), three non-typhoidal Salmonella (Salmonella choleraesuis CMCC50018, Salmonella pullorum ATCC10398, Salmonella enteritidis ATCC13076), and three non-Salmonella strains (Staphylococcus aureus CICC26003, Escherichia coli O157:H7 ATCC43889, Listeria monocytogenes CICC21635, Klebsiella pneumoniae ATCC46117). All Salmonella strains were grown in fresh TSB medium and cultured at 37°C. Listeria monocytogenes was grown in TSB-YE medium, and the other bacterial strains were cultured in fresh LB medium at 37°C.

[0037] BALB / c mice and Kunming mice were purchased from Shanghai JSG Biotechnology Co., Ltd. SP2 / 0 myeloma cells were maintained in our laboratory.

[0038] Prime STAR DNA polymerase, restriction enzymes, and gel extraction kits were purchased from TaKaRa; plasmid extraction kits were purchased from Tiangen; kanamycin (Kan) was purchased from Invitrogen; fetal bovine serum (FBS) and Dulbecco's Membrane Medium (DMEM) were purchased from Invitrogen (Shanghai) Trading Co., Ltd. Freund's adjuvant, polyethylene glycol (PEG 6000), hypoxanthine (H), aminopterin (A), thymidine (T), penicillin (PS), and L-glutamine (LG) were all products of Sigma. Skim milk powder was purchased from Sangon Biotechnology Co., Ltd.; serum-free cell freezing medium was purchased from Suzhou Xinsaimei Biotechnology Co., Ltd.; and antibody subtype identification kits were purchased from Southern Biotech Co., Ltd. Trizol reagent was a product of Invitrogen; Prime Script DNA-free reverse transcription kits and are products of Thermo Biotechnology Co., Ltd.

[0039] HT stock solution (100×): Dissolve 38.8 mg of thymidine and 136.1 mg of hypoxanthine in 50 mL of ddH2O and dilute to 100 mL. Place in a 45°C water bath to completely dissolve. Filter through a 0.22 μm filter membrane and aliquot. Store at -20°C until ready for use.

[0040] Stock solution A (100×): Dissolve 1.76 mg of aminopterin in appropriate amount of dd H2O, add 1 mL of 1 mol / L NaOH, and dilute to 100 mL with dd H2O. Filter through a 0.22 μm filter membrane, aliquot, and store at -20°C until needed.

[0041] 20% FBS culture medium: 1 mL penicillin-streptomycin (PS), 1 mL L-glutamine (LG), 20 mL fetal bovine serum (FBS) and 78 mL DMEM, stored at 4°C until use.

[0042] HT medium: 1 mL LG, 1 mL PS, 1 mL HT, 20 mL FBS, and 77 mL DMEM. Store at 4°C until use.

[0043] HAT medium: 1 mL A, 1 mL HT, 1 mL LG, 1 mL PS, 20 mL FBS, and 76 mL DMEM. Store at 4°C until use.

[0044] ELISA coating solution (pH 9.6): Add 0.848 g Na2CO3 and 1.428 g NaHCO3 to ddH2O and adjust the volume to 0.5 L. Store at 4°C.

[0045] ELISA substrate colorimetric solution: Solution A is prepared by adding 6 mL of glacial acetic acid, 2.45 g of sodium acetate, and 0.3 mL of 30% hydrogen peroxide to 500 mL in ddH2O. Solution B is prepared by adding 0.2 g of disodium EDTA, 0.95 g of citric acid, 0.15 g of TMB, and 50 mL of glycerol to 500 mL in ddH2O. When using, mix Solution A and Solution B in a 1:1 ratio (prepare immediately before use).

[0046] ELISA blocking solution (5% skim milk): 5 g skim milk was dissolved in PBST and the volume was adjusted to 100 mL.

[0047] ELISA stop solution (2M H2SO4): Slowly add 89 mL ddH2O to 11 mL 98% concentrated sulfuric acid while stirring and store at 4°C.

[0048] ELISA wash buffer (1×PBST, pH 7.2): 8.0 g NaCl, 2.9 g Na2HPO4·12H2O, 0.2 g KH2PO4, and 0.2 g KCl were added to ddH2O to make up to 1000 mL, and after dissolution, 0.5 mL Tween-20 was added.

[0049] The emulsifier was purchased from Amalgamator, the desktop low-temperature centrifuge was purchased from Thermo Fisher Technology Co., Ltd.; the CO2 cell culture incubator was purchased from Thermo Scientific, USA; the multifunctional microplate reader was purchased from BIOTEK, USA; and the gel imaging system was purchased from Bio-Rad China.

[0050] LB liquid medium: 10 g NaCl, 5 g yeast extract, and 10 g tryptone were diluted to 1 L of deionized water, the pH was adjusted to 7.4, autoclaved at 121°C for 15 min, and stored at 4°C until used. LB solid medium: 10 g NaCl, 5 g yeast extract, 10 g tryptone, and 15 g agar powder were diluted to 1 L of deionized water, the pH was adjusted to 7.4, autoclaved at 121°C for 15 min, and stored at 4°C after plate preparation. BHI liquid medium, TSB liquid medium, and THB liquid medium were purchased from BD Biotechnology. Salmonella typhimurium monoclonal antibody 7H9 was prepared previously in our laboratory. 2-(N-Morpholino)ethanesulfonic acid and 4-Morpholinoethanesulfonic acid (MES) were purchased from SIGMA, Cat. No. M3671-50G. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl): Purchased from Thermo Scientific, Catalog No. PA184225. N-Hydroxyphthalimide (NHS): Purchased from SIGMA. Activation buffer (MEST): Dissolve 0.488 g of MES in 100 mL of ddH2O, adjust the pH to 6.0 with NaOH, add 50 μL of Tween-20, and filter through a 0.22 μm filter. Store at 4°C. Coupling buffer (MEST): Dissolve 0.488 g of MES in 100 mL of ddH2O, adjust the pH to 7.0 with NaOH, add 50 μL of Tween-20, and filter through a 0.22 μm filter. Store at 4°C. EDC solution: Prepare 5 mg / mL EDC (25 mM, pH 6.0) from MES stored at 4°C and filter through a 0.22 μm filter. NHS solution: 5 mg / mL NHS (25 mM, pH 6.0) was prepared from NHS stored at 4°C and filtered through a 0.22 μm filter. Prepare for immediate use. Blocking solution: 1% BSA in PBST (pH 7.4), filtered through a 0.22 μm filter. Store at 4°C. Storage solution: 0.2 g NaN3 and 5 g BSA, dissolved in 1000 mL 0.01 M PBST (pH 7.4), then filtered through a 0.22 μm filter. Store at 4°C. Fresh milk was collected from a dairy farm in Shanghai. PM3-020 (180 nm in diameter), PM3-050 (750 nm in diameter), SM3-P100 (1150 nm in diameter), and 70102-5 (2000 nm in diameter) superparamagnetic nanoparticles were purchased from Shanghai Aorun Micro-Nano New Material Technology Co., Ltd.

[0051] Example 1 Preparation of Salmonella typhimurium immunogen

[0052] Salmonella typhimurium was cultured to OD 600When the value is 1.0, add formaldehyde to a final concentration of 0.3% and inactivate the cells at 37°C in a shaker at 200 rpm for 4 hours. Then, take 1 mL of each bacterial solution and place it in a 1.5 mL EP tube. Centrifuge at 12,000 rpm for 5 minutes, discard the supernatant, and wash the pellet five times with PBS. Then, mix equal amounts of the three bacterial solutions and adjust the concentration to 5.0 × 10 9 CFU / mL.

[0053] Example 2 Animal Immunization Program

[0054] The primary immunization consisted of a bacterial solution and an equal volume of Freund's complete adjuvant. Immunizations for the second, third, fourth, and fourth immunizations consisted of a bacterial solution and an equal volume of Freund's incomplete adjuvant, emulsified for 90 seconds before immunization. For the fifth intraperitoneal immunization, the bacterial solution was diluted with saline. The immunization schedule for mice is shown in Table 1.

[0055] Table 1 Mouse immunization program

[0056]

[0057] Example 3 Animal Serum Titer Determination

[0058] Balb / C mice were immunized, and mouse serum was collected after the fourth immunization at a ratio of 1:2×10 3 , 1:4×10 3 , 1:8×10 3 , 1:16×10 3 1:32×10 3 , 1:64×10 3 , 1:128×10 3 and 1:256×10 3 After proportional dilution, the serum titer was determined by indirect ELISA, and healthy mouse serum was used as a negative control. The best immune mice were selected for cell fusion.

[0059] Indirect ELISA experimental steps:

[0060] (1) Dilute the inactivated Salmonella typhimurium (mixed in equal proportions) with ELISA coating solution to 5.0×10 8 Add 100 μL / well of the diluted coating solution to a 96-well microtiter plate. Use a blank control without the coating agent and incubate at 37°C for 4 hours or at 4°C overnight.

[0061] (2) After coating, discard the coating solution and wash the ELISA plate three times with washing buffer. Add 5% skim milk (200 μL / well) to each well and place in a 37°C incubator for 2 h.

[0062] (3) After blocking, wash the plate three times with washing buffer to obtain the Salmonella typhimurium antibody ELISA test plate, which is then stored at 4°C for future use.

[0063] (4) The serum to be tested was serially diluted with 5% FBS in PBST (1×PBST, pH 7.2), and the serially diluted serum was added to the Salmonella typhimurium antibody ELISA test plate (100 μL per well), and incubated at 37°C for 2 h.

[0064] (5) Wash the Salmonella typhimurium antibody ELISA test plate three times with washing buffer, add 100 μL of diluted HRP-labeled goat anti-mouse IgG antibody (1:2000) to each well, and incubate at 37°C for 1 hour.

[0065] (6) After incubation, wash three times with washing buffer, add 100 μL of color development solution to each well, incubate at 37°C for 15 minutes, add 50 μL of stop solution, and finally read the OD using a microplate reader. 450 The absorbance value below.

[0066] The mice were bled on the 7th to 10th day after the 4 immunizations, and the serum titers of the mice were determined using the above-mentioned indirect ELISA method. The experimental results showed that the serum titers of the mice were all around 256×10 3 The titers were all high and suitable for cell fusion. The mice with the highest titers were selected for booster immunization, i.e., the immunogen was reduced by half for intraperitoneal impact immunization.

[0067] Table 2 ELISA detection of serum levels in mice after immunization

[0068]

[0069] Example 4 Preparation of Salmonella typhimurium monoclonal antibodies

[0070] 1. Cultivation of myeloma cells (SP2 / 0)

[0071] Remove the SP2 / 0 cells from the liquid nitrogen tank and quickly place them in a 37°C waterbath until completely thawed. Add 7 mL of pre-prepared blank DMEM medium to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 8 minutes. Discard the supernatant and resuspend the SP / 20 cells in an appropriate amount of complete DMEM medium. Transfer the cells to a cell culture flask and culture in a 37°C, 5% CO2 incubator. Observe cell growth daily and change the medium promptly. Split the cells when they reach 90% of the bottom of the flask. To ensure optimal cell growth at fusion, replace the cells with fresh medium the evening before fusion. Remove the SP2 / 0 cells from the liquid nitrogen tank and quickly place them in a 37°C waterbath until completely thawed. Add 7 mL of pre-prepared blank DMEM medium to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 8 minutes. Discard the supernatant and resuspend the SP / 20 cells in an appropriate amount of complete DMEM medium. Transfer the cells to a cell culture flask and culture in a 37°C, 5% CO2 incubator. Observe the cell growth status daily and replace the culture medium in time. When the cells grow to 90% of the bottom surface of the culture flask, split the cells into flasks. To ensure the cells are in the best growth state at the time of fusion, replace the cells with fresh culture medium one night before fusion.

[0072] 2. Preparation of Feeder Cells

[0073] 6-8 week-old Kunming mice were sacrificed by exsanguination and immersed in 75% alcohol for 5 minutes. After disinfection, the mice were fixed in a clean bench with their abdomen facing upward. Using autoclaved scissors and forceps, the abdominal skin was incised along the linea alba to expose the peritoneum, taking care not to touch the peritoneum. Using a 5mL syringe, 4mL of DMEM medium was injected into the mouse's peritoneal cavity. The peritoneal cavity was then gently tapped approximately 50 times with forceps. The medium in the peritoneal cavity was then withdrawn from the needle using the same syringe and transferred to a 15mL centrifuge tube. This procedure was repeated 2-3 times. The collected peritoneal lavage fluid was transferred to a sterile centrifuge tube and centrifuged at low speed for 10 minutes. The supernatant was discarded, and the feeder cells were resuspended in 50mL of HAT medium. The feeder cells were then added dropwise to a 96-well cell culture plate and placed in a 5% CO2 cell culture incubator until ready for use.

[0074] 3. Cell Fusion

[0075] (1) Preparation of SP2 / 0 cells: Collect well-growing SP2 / 0 cells in a centrifuge tube, centrifuge at 1000 rpm for 8 min, discard the supernatant, resuspend the cells and transfer them to a fusion tube.

[0076] (2) Isolation of spleen cells: The mice to be used for fusion were euthanized, and then the abdominal skin and peritoneum of the mice were cut open in sequence according to the above-mentioned operation method to fully expose the internal organs. On the left side of the mouse internal organs, the fat and other connective tissues adhering to the spleen were separated, and the spleen was removed and placed in a 12-well cell culture plate. The surface was washed with blank DMEM medium and then transferred to another well. The spleen was repeatedly rinsed with blank DMEM medium using a syringe until the spleen cells were washed until transparent. The eluate was centrifuged at 1000 rpm for 8 minutes and the supernatant was discarded. Then the supernatant was discarded and the plate was resuspended for later use.

[0077] (3) Fusion of SP2 / 0 and spleen cells: After mixing, transfer the mixture to a fusion tube containing SP2 / 0 cells, centrifuge at 1000 rpm for 8 minutes, and completely discard the supernatant. Place the fusion tube on the palm of your left hand and pat it. After vigorous shaking, mix the two cells thoroughly and place it in a 37°C incubator for 10 minutes. Add 1 mL of preheated PEG evenly within 45 seconds. After 90 seconds, add an appropriate amount of DMEM medium to terminate the PEG reaction. Then, incubate the fusion tube at 37°C for 10 minutes, centrifuge at 1000 rpm for 8 minutes, discard the supernatant, and resuspend it in an appropriate amount of 20% FBS HAT medium. Dilute it to different concentrations and add it to a 96-well plate with feeder cells (100 μL / well). Culture it in a 37°C, 5% CO2 incubator.

[0078] 4. Screening of Positive Cells

[0079] Observe the growth status daily. Screening for positive cells depends on the growth status of the hybridoma cells. Observe the cell growth status 8-10 days after fusion. Screen and mark the wells where only monoclonal cell clusters grow. Take 100 μL of the supernatant from each monoclonal well and replenish HAT medium. Use the indirect ELISA method to determine the Salmonella typhimurium solution. The coating buffer is diluted to 5.0×10 8 CFU / mL was coated on a 96-well ELISA plate. Meanwhile, negative mouse serum was used as a negative control, and blank coating buffer was used as a blank control. An appropriate amount of cell supernatant and PBST (containing 5% FBS) were mixed to make 100 μL as the primary antibody. The remaining ELISA procedures were the same as those for the mouse serum titer determination, and positive wells were identified.

[0080] 5. Subcloning of Positive Hybridoma Cells

[0081] Pick monoclonal cell lines with high titer of cell supernatant and good growth status for subcloning. First, mark the cell position of the positive clone under a microscope, use a 200μL pipette to draw a small amount of cells and transfer them to a 96-well cell plate, and perform gradient dilution with 20% FBS incomplete culture medium. Observe under an inverted microscope, select wells containing 90-120 cells per well, transfer the liquid in the well to 10mL 20% FBS incomplete culture medium, and mix well by pipetting. Then add dropwise to a 96-well cell plate containing feeder cells (100μ / well) and culture in a 37°C, 5% CO2 incubator. Observe the cell growth status in time and supplement the culture medium in an appropriate amount within 3-5 days. When the cells grow to the size of a fingernail, perform screening and subcloning again until the positive rate of the cell supernatant of the monoclonal well reaches 100%, expand the culture and freeze while marking.

[0082] On days 3-4 after cell fusion, examine all cell culture wells under a microscope and mark the 96-well plates containing one or two monoclonal clones. On days 8-9 after cell fusion, select the culture supernatant containing monoclonal clones for titer testing. Subsequently, select monoclonal cell lines with high titer and good specificity for subcloning.

[0083] 6. Preparation of Mouse Ascites

[0084] Female Balb / C mice were intraperitoneally injected with 0.5 mL of sterilized paraffin oil every 7 days for a total of 3 injections. Then, 0.5 mL of subcloned positive cells (concentration of 2×10 6 / L) and an equal volume of sterile paraffin oil. Observe the physiological and mental state of the mice several times daily for 7-15 days after injection. For mice that are lethargic, have a distended abdomen, or experience fluctuations when pressed, collect ascites with a sterile syringe needle when they are on the verge of death. After the ascites has settled for 1-2 hours, centrifuge at 4°C and 5000 rpm for 30 minutes. After centrifugation, the ascites separates into three layers, the middle layer of which is the yellow, translucent ascites. In a clean bench, collect the ascites with a pipette, store in clean EP tubes, and freeze at -80°C.

[0085] Example 5 Monoclonal Antibody Analysis

[0086] 1. Potency and Specificity Determination

[0087] The titer of Salmonella typhimurium-specific monoclonal antibodies was measured using the ELISA method. Three inactivated Salmonella typhimurium bacterial solutions were mixed using coating buffer, and 100 μL per well was coated on a 96-well ELISA plate. The antibody detection steps were the same as in Example 3. The collected ascites monoclonal antibodies were serially diluted at 1:10,000, 1:20,000, 1:40,000, 1:80,000, 1:160,000, 1:320,000, 1:640,000, 1:1,280,000, 1:2,560,000, 1:5,120,000, 1:10,240,000, 1:20,480,000, and 1:40,960,000. 100 μL of each concentration was added to each well as the primary antibody. The remaining ELISA operation steps were the same as those for the mouse serum titer determination. At the same time, set up positive and negative controls. Measure the OD value using a microplate reader. 450 The titer was determined by a P / N value ≥ 2.1, and the maximum dilution was the titer of the antibody. Inactivated Salmonella choleraesuis CMCC50018, Salmonella pullorum ATCC10398, Salmonella enteritidis ATCC13076, Staphylococcus aureus CICC26003, Escherichia coli O157:H7 ATCC43889, Listeria monocytogenes CICC21635, and Klebsiella pneumoniae ATCC46117 were coated on a 96-well ELISA plate using coating buffer. The antibody detection procedure was the same as in Example 3 to determine the specificity of the monoclonal antibody.

[0088] After 4 subcloning, two monoclonal antibodies were screened and named 7H9 and 2D10. The titer of ascites was determined by indirect ELISA using Salmonella typhimurium as the coating source. The specific method is the same as above. As can be seen from the results (Table 3), the titers of the two monoclonal antibodies are high. When diluted 5120000 times, the OD 450 The titers remained above 0.6, with a P / N value ≥ 2.1, indicating that the titers of 7H9 and 2D10 were 1:20,480,000 and 1:10,240,000, respectively. The specificity of the antibodies was determined by diluting 7H9 and 2D10 50,000-fold using inactivated Salmonella choleraesuis CMCC50018, Salmonella pullorum ATCC10398, Salmonella enteritidis ATCC13076, Staphylococcus aureus CICC26003, Escherichia coli O157:H7 ATCC43889, Listeria monocytogenes CICC21635, and Klebsiella pneumoniae ATCC46117 as coating agents. The results (Table 4) showed that neither antibody reacted with the eight common foodborne pathogens, indicating that both antibodies had good specificity.

[0089] Table 3 OD of monoclonal antibodies 450 Determination

[0090] Dilution multiple 7H9 2D10 1:10,000 3.58 3.22 1:20,000 3.46 3.19 1:40,000 3.17 2.83 1:80,000 2.85 2.42 1:160,000 2.85 2.51 1:320,000 2.01 1.87 1:640,000 1.76 1.53 1:1,280,000 1.39 1.07 1:2,560,000 1.11 0.89 1:5,120,000 0.77 0.62 1:10,240,000 0.42 0.38 1:20,480,000 0.25 0.11 1:40,960,000 0.15 0.10 Negative 0.07 0.07

[0091] Table 4 Specificity determination of monoclonal antibodies

[0092] 50,000-fold dilution of monoclonal antibody 7H9 2D10 Salmonella choleraesuis CMCC50018 0.13 0.18 Salmonella pullorum ATCC10398 0.12 0.14 Salmonella Enteritidis ATCC13076 0.36 0.42 Escherichia coli O157ATCC43889 0.11 0.14 Listeria monocytogenes CICC21635 0.12 0.14 Klebsiella pneumoniae ATCC46117 0.10 0.14 Negative 0.15 0.11

[0093] 2. Identification of Monoclonal Antibodies

[0094] A 96-well ELISA plate was coated with inactivated Salmonella typhimurium, and the two positive antibodies collected were identified by antibody subtype using a monoclonal antibody subtype identification kit. The primary antibody was diluted at a multiple of 1:10,000, and the secondary antibody in the kit was added at a dilution multiple of 1:300.

[0095] The results of subtype identification of the two monoclonal antibodies using an antibody subtype identification kit showed that the light chain type of both antibodies was kappa type and the heavy chain type was IgG1 type.

[0096] Table 5 Isotype determination of monoclonal antibodies

[0097] 7H9 2D10 light chain Kappa Kappa Heavy chain IgG1 IgG1

[0098] Example 6 Determination and Analysis of Anti-Salmonella Typhimurium Monoclonal Antibody Sequences

[0099] The monoclonal antibody 7H9 has the best potency and specificity. In order to identify the 7H9 antibody sequence, total RNA was isolated from hybridoma cells secreting the monoclonal antibody 7H9 using TRIzol reagent. Specific steps: 1) Trizol lysis: Resuspend all bacteria with 1 mL of Trizol, mix thoroughly by pipetting, and let stand for 3-5 minutes. (2) Chloroform phase separation: Add 0.2 mL of chloroform (trichloromethane) to each sample, shake vigorously for 15 seconds, let it sit at room temperature for 3 minutes, and then centrifuge at 12,000 rpm for 15 minutes. (3) RNA precipitation: Transfer the upper aqueous phase to a new 1.5 mL RNase-free EP tube, add 500 μL of isopropanol to precipitate the RNA molecules, and mix thoroughly by inversion. (4) RNA washing: Gently aspirate the supernatant (be careful not to aspirate the white precipitate), drain on paper, add 1 mL of 75% ethanol (750 μL of anhydrous ethanol, 250 μL of DEPC water, prepared in advance) to the precipitate, and centrifuge at 7,500 rpm for 5 minutes. (5) RNA dissolution: Discard the supernatant and dry for 10 minutes. Add appropriate amount of DEPC water to dissolve, place on ice, and measure OD 260 / OD 280 .

[0100] The antibody constant region-specific primers were then used to reverse transcribe the fragments into cDNA using a Thermo reverse transcription kit. PCR amplification was performed using the corresponding cDNA as a template using Ex Taq enzyme (Takara) and degenerate primers, followed by sequencing to obtain the variable region sequences of the heavy and light chains of the monoclonal antibody. The locations of the complementary determining regions (CDRs) were determined using the IgBLAST tool.

[0101] To determine the antibody sequence, RNA was extracted from hybridoma cells and the variable region sequence was amplified using degenerate primers. The sequencing results showed that the sequence of the monoclonal antibody variable region was a gene fragment of the germline gene family (Table 6).

[0102] Table 6 Germline genes in the variable regions of anti-Salmonella typhimurium monoclonal antibodies (IgBlast analysis)

[0103] MAb <![CDATA[V H ]]> <![CDATA[D H ]]> <![CDATA[J H ]]> <![CDATA[V K ]]> <![CDATA[J K ]]> 7H9 5-9*04 1-1*01 3*01 3-12*01 1*01

[0104] Note: V H , D H , J H They are the V, D, and J germline gene segments of the heavy chain variable region. K , J K They are the V and J germline gene segments of the light chain variable region, respectively.

[0105] The sequencing results are as follows:

[0106] Light chain nucleotide sequence (SEQ ID NO: 10):

[0107]

[0108]

[0109] Note: The bold part is the variable region nucleotide sequence, and the non-bold part is the constant region nucleotide sequence.

[0110] Light chain amino acid sequence (SEQ ID NO: 7):

[0111]

[0112] Note: The bold part is the variable region amino acid sequence, and the non-bold part is the constant region amino acid sequence. The bold and underlined parts are the complementarity determining regions (CDRs).

[0113] in:

[0114] LCDR1:KSVSTSGSSY(SEQ ID NO:1)

[0115] LCDR2: LAS

[0116] LCDR3:QHSGVLTWT(SEQ ID NO:2)

[0117] Light chain variable region amino acid sequence (SEQ ID NO: 6):

[0118] DIVLTQSPASLAVSLGQRATISCRASKSVSTSGSSYMHWYQQKPGQPPKLLIYLASNLESGVDIVLTQSPASLAVSLGQRATISCRASKSVSTSGSSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSGVLTWTFGGGTKLEIK

[0119] Heavy chain nucleotide sequence (SEQ ID NO: 11):

[0120]

[0121]

[0122] Note: The bold part is the variable region nucleotide sequence, and the non-bold part is the constant region nucleotide sequence. Heavy chain amino acid sequence (SEQ ID NO: 9):

[0123]

[0124]

[0125] Note: The bold part is the variable region amino acid sequence, and the non-bold part is the constant region amino acid sequence. The bold and underlined parts are the complementarity determining regions (CDRs).

[0126] in:

[0127] HCDR1: GFTFSSYS (SEQ ID NO: 3)

[0128] HCDR2: ISGFGRNT (SEQ ID NO: 4)

[0129] HCDR3: ARRGSSAY (SEQ ID NO: 5)

[0130] Heavy chain variable region amino acid sequence (SEQ ID NO: 8):

[0131] EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYSMSWVRQTPEKRLEWVATISGFGRNTYYPDSVKGRFSSSRDNANNTLYLQMSSLRSEDTAIYYCARRGSSAYWGRGTLVTVSA

[0132] Example 7 Preparation of Salmonella typhimurium immunomagnetic beads

[0133] 1. Activation of Immunomagnetic Beads

[0134] (1) Take 2 mg (i.e., 200 μL, 10 mg / mL) of magnetic beads and place them in a 1.5 mL EP tube. Place the tube on a magnetic rack. Let it stand for 1 min. After the magnetic beads and supernatant are separated, discard the supernatant. Add 500 μL of activation buffer (MEST, pH 6.0) to resuspend the magnetic beads. Vortex the tube to mix evenly. Place the tube on a magnetic separation rack for 1 min. Discard the supernatant. Repeat the washing process 3 times, 1 min each time, and discard the supernatant.

[0135] (2) Add 200 μL EDC (5 mg / mL) and 200 μL NHS (5 mg / mL) (prepared immediately before use), mix the magnetic beads, and place them on a shaker at 37°C, 200 rpm / min, for activation for 30 min;

[0136] (3) Remove, spin briefly, place on a magnetic stand for 1 min, add 500 μL activation buffer (MEST, pH 6.0), mix the magnetic beads, and transfer them to a new EP tube;

[0137] (4) Magnetic separation: remove the supernatant, wash twice with 500 μL activation buffer (MEST, pH 6.0), perform magnetic separation using a magnetic stand, and aspirate the supernatant with a pipette. At this point, the carboxyl groups on the surface of the magnetic beads have been activated and can be covalently coupled with biological ligands with primary amino groups.

[0138] 2. Immunomagnetic bead labeling of monoclonal antibodies

[0139] (1) Wash once with 500 μL coupling buffer MEST (MEST, pH 7.0) and discard the supernatant;

[0140] (2) Take a new EP tube, add 500 μL of coupling buffer MEST (MEST, pH 7.0), draw an appropriate amount of monoclonal antibody 7H9 and the coupling buffer, vortex and shake, and then aspirate the mixture into the EP tube containing magnetic beads. After mixing, place it in a rotating incubator at 30 rpm / min and react at room temperature for at least 3 h;

[0141] (3) After coupling, gently centrifuge, place the EP tube on a magnetic separation rack, let it stand for 1 min, and gently wash three times with 500 μL PBST;

[0142] (4) Add 1 mL of blocking solution for blocking, and place the EP tube in a 37°C shaker at 200 rpm / min for at least 30 minutes (or overnight at 4°C);

[0143] (5) Remove the tube, spin gently and briefly, place it on a magnetic rack, let it stand, discard the supernatant, and wash it 4 times with PBST, 1 min each time, and discard the supernatant;

[0144] (6) Add 600 μL of storage solution, resuspend the immunomagnetic beads, and store at 4°C until use.

[0145] Example 8 Immunomagnetic Bead Detection Process

[0146] The culture conditions for each bacterium were 37℃ and 180rpm shaking. 600 When it is 1.0, use PBST to perform a 10-fold (or 5-fold) gradient dilution of the bacterial solution until it reaches the appropriate concentration. At this time, aspirate an appropriate amount of diluted bacterial solution and add it to a new EP tube, take an appropriate amount of coupled immunomagnetic beads (gently mix in advance) and mix it, place it on a rotating incubator, 30r / min, and react at room temperature for a certain time. At the same time, aspirate an appropriate amount of diluted bacterial solution to apply to the LB plate and calculate the concentration of the original bacterial solution. After the capture is completed, gently separate and place it on a magnetic stand. Aspirate 100μL of the supernatant, apply it to the LB plate, and calculate the number of residual bacteria in the supernatant after the magnetic beads are captured. Then gently wash the immunomagnetic beads once with 500μL PBST, resuspend the magnetic beads with an appropriate amount of PBST and mix them, aspirate 100μL and hang it on the LB plate, and calculate the number of magnetic beads captured. The formula for calculating the capture rate of immunomagnetic beads is as follows:

[0147] Capture rate (%) = number of bacteria captured by immunomagnetic beads / (number of bacteria in supernatant + number of bacteria captured by immunomagnetic beads) × 100%

[0148] Example 9 Optimization of immunomagnetic bead detection conditions

[0149] 1. Determination of the Optimal Diameter of Immunomagnetic Beads

[0150] According to previous studies, changes in the diameter of immunomagnetic beads can have a significant impact on the capture of specific bacteria. In this experiment, Salmonella typhimurium ATCC14028 was used as the target bacteria, and PBST gradient dilution was performed to about 10 4 CFU / mL. Four different sizes of magnetic beads from Orun Co., Ltd.—PM3-020 (180 nm diameter), PM3-050 (750 nm diameter), SM3-P100 (1150 nm diameter), and 70102-5 (2000 nm diameter)—were used for coupling and capture experiments, following the same methods as above. This experiment was repeated three times, and the capture efficiency was calculated to determine the optimal diameter of the immunomagnetic beads.

[0151] The immunomagnetic beads prepared with different particle sizes have different effects. In this experiment, magnetic beads with particle sizes of 180, 750, 1150, and 2000 nm were used to prepare immunomagnetic beads to capture the concentration of 5×10 4 The capture rates of Salmonella typhimurium with the four sizes of magnetic beads were 95.23%, 87.6%, 88.92% and 79.55% respectively. The highest capture rate was achieved with the 180nm diameter magnetic beads, so the subsequent experiments selected the 180nm diameter magnetic beads ( Figure 1 ).

[0152] 2. Determination of the Optimal Amount of Immunomagnetic Beads

[0153] To explore the effect of different amounts of immunomagnetic beads on the capture rate, Salmonella typhimurium ATCC14028 was used as the target bacteria and the PBST gradient was diluted to about 10 4 CFU / mL, using immunomagnetic beads for capture experiments. The optimal working dosage of immunomagnetic beads was determined by adding 0.1, 0.2, 0.3, 0.4, and 0.5 mg of immunomagnetic beads to each tube in a 1 mL reaction system. This experiment was repeated three times, and the capture rate was calculated to determine the optimal number of immunomagnetic beads.

[0154] like Figure 2 As shown in the figure, when the usage is less than 0.3 mg, the capture rate increases with the increase of magnetic bead dosage; after the magnetic bead dosage exceeds 0.3 mg, the capture rate tends to increase slowly. In order to achieve the maximum capture rate, the optimal magnetic bead usage is 0.4 mg, and the capture rate can reach 97.73%.

[0155] 3. Determination of the Optimal Capture Time for Immunomagnetic Beads

[0156] To explore the effect of different capture time on the capture rate, the target bacteria were Salmonella typhimurium ATCC14028, and the PBST gradient was diluted to about 10 4 CFU / mL, using immunomagnetic beads for capture experiments. Capture was performed at detection times of 10, 20, 30, 40, 50, and 60 minutes. This experiment was repeated three times, and the capture rate was calculated to determine the optimal capture time.

[0157] like Figure 3 As shown, the optimal capture time for immunomagnetic beads was determined using five time gradients: 15, 30, 45, 60, and 75 minutes. Between 15 and 45 minutes, the capture rate increased with increasing time, exceeding 90% after 45 minutes. For rapid enrichment, the optimal capture time for this experiment was 45 minutes.

[0158] 4. Specificity testing of immunomagnetic bead detection method

[0159] The specificity of the immunomagnetic beads was tested using the same experimental methods as above using strains of Salmonella Typhimurium ATCC14028, ATCC21484, and ATCC10420, Staphylococcus aureus CMCC26003, Listeria monocytogenes CICC21635, Enterohemorrhagic Escherichia coli O157 ATCC43889, Vibrio parahaemolyticus SH112, and Klebsiella pneumoniae ATCC46117. This experiment was repeated three times, and the capture rate was calculated to confirm the specificity of the immunomagnetic beads.

[0160] like Figure 4 As shown, the prepared immunomagnetic beads achieved capture rates exceeding 95% for three strains of Salmonella Typhimurium (ATCC14028, ATCC21484, and ATCC10420): 96.89%, 97.06%, and 95.41%, respectively. Capture rates for other common foodborne strains were all below 5%, including 1.81% for Staphylococcus aureus (CMCC26003), 0.83% for Listeria monocytogenes (CICC21635), 4.43% for Enterohemorrhagic Escherichia coli (ATCC43889), 0.62% for Vibrio parahaemolyticus (SH112), and 2.57% for Klebsiella pneumoniae (ATCC46117). This demonstrates the high specificity of the prepared immunomagnetic beads.

[0161] 5. Sensitivity Determination of Immunomagnetic Bead Detection Method

[0162] To explore the effect of different bacterial concentrations on the capture rate, the target bacteria were Salmonella typhimurium ATCC14028, and the PBST gradient was diluted to about 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 CFU / mL. Capture experiments were performed using immunomagnetic beads. The experiments were repeated three times and the capture efficiency was calculated.

[0163] like Figure 5 , the target bacteria in the capture system is as low as 10 2 CFU / mL, the capture rate of the immunomagnetic beads can still reach more than 90%, indicating that the immunomagnetic beads have good sensitivity.

[0164] 6. Immunomagnetic Bead Enrichment of Salmonella Typhimurium in Milk Samples

[0165] Dilute the bacterial solution to 1×10 4 , 1×10 3 and 1×10 2CFU / mL, and added to simulated clinical samples of pasteurized milk purchased from supermarkets. The capture system was 100 μL of magnetic beads added to 900 μL of simulated milk sample, mixed evenly, placed on a vortex incubator, and captured at 30 rpm and room temperature for 45 minutes. The rest of the steps were the same as above. The capture rate was calculated to determine the capture performance of Salmonella typhimurium immunomagnetic beads in milk. 4 CFU / mL of Salmonella typhimurium in PBST buffer was used as a positive control.

[0166] Salmonella typhimurium was added to sterilized milk and the prepared immunomagnetic beads were used to enrich Salmonella typhimurium. The results were as follows: Figure 6 PBST capture system was used as a control. 4 CFU / mL of Salmonella typhimurium, the capture rate of the immunomagnetic beads reached 98.62%; when the milk contained 10 2 CFU / mL, 10 3 CFU / mL, 10 4 When the CFU / mL of Salmonella typhimurium was 0.05, the capture rates were 85.42%, 89.73% and 92.15% respectively, indicating that the capture rate of the immunomagnetic beads in milk samples can still reach a high level of more than 85%, which has good practical application value.

Claims

1. A monoclonal antibody against Salmonella typhimurium, comprising a light chain variable region and a heavy chain variable region, characterized in that: The amino acid sequence of LCDR1 in the light chain variable region is shown in SEQ ID NO: 1, the amino acid sequence of LCDR2 is LAS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 2; the amino acid sequences of HCDR1 to HCDR3 in the heavy chain variable region are shown in SEQ ID NOs: 3, 4 and 5, respectively.

2. The monoclonal antibody according to claim 1, wherein The light chain variable region comprises the sequence shown in SEQ ID NO: 6, and the heavy chain variable region comprises the sequence shown in SEQ ID NO:

8.

3. The monoclonal antibody according to claim 2, wherein The light chain of the monoclonal antibody comprises the sequence shown in SEQ ID NO: 7, and the heavy chain comprises the sequence shown in SEQ ID NO:

9.

4. A polynucleotide, characterized in that It encodes the monoclonal antibody according to any one of claims 1 to 3.

5. The polynucleotide according to claim 4, wherein The polynucleotide comprises a light chain nucleotide as shown in SEQ ID NO: 10 and / or a heavy chain nucleotide as shown in SEQ ID NO:

11.

6. A recombinant expression vector, characterized in that: It comprises the polynucleotide according to claim 4 or 5.

7. A transformant, characterized in that: It comprises the polynucleotide according to claim 4 or 5 or the recombinant expression vector according to claim 6.

8. The transformant according to claim 7, wherein The host is Escherichia coli.

9. An antibody-labeled immunomagnetic bead, characterized in that The antibody is a monoclonal antibody according to any one of claims 1 to 3.

10. The immunomagnetic beads according to claim 9, wherein The immunomagnetic beads are activated during preparation.

11. The immunomagnetic beads according to claim 10, wherein The diameter of the immunomagnetic beads is 180-2000 nm.

12. The immunomagnetic beads according to claim 10, wherein The activation method comprises the following steps: (1) Mix the immunomagnetic beads with the MEST solution; (2) Add a mixed solution of EDC and NHS to obtain activated immunomagnetic beads.

13. The immunomagnetic beads according to claim 12, wherein The pH of the MEST solution is 6.

14. The immunomagnetic beads according to claim 11, wherein The diameter of the immunomagnetic beads is 180 nm.

15. The immunomagnetic beads according to claim 12 or 13, wherein The volume ratio of EDC and NHS in step (2) is 1:1, and the concentration of both is 5 mg / mL.

16. A method for enriching and isolating Salmonella typhimurium, characterized in that: The immunomagnetic beads according to any one of claims 9 to 15 are used to enrich and separate Salmonella typhimurium.

17. The method according to claim 16, wherein The enrichment comprises the following steps: (1) Incubating the immunomagnetic beads with the sample to be tested in coupling buffer for 45 to 75 minutes; Optionally, the method further includes (2) calculating a capture rate.

18. The method according to claim 17, wherein The volume of the enriched reaction system and the relative usage of the immunomagnetic beads are 1 mL: 0.1-0.5 mg.

19. The method according to claim 18, wherein The volume of the enriched reaction system and the relative usage of the immunomagnetic beads are 1 mL: 0.3-0.5 mg.

20. The method of claim 18, wherein: The volume of the enriched reaction system and the relative usage of the immunomagnetic beads were 1 mL:0.4 mg.

21. Use of the monoclonal antibody according to any one of claims 1 to 3 or the immunomagnetic beads according to any one of claims 9 to 15 in enriching and separating Salmonella typhimurium or preparing a reagent for enriching and separating Salmonella typhimurium.

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