Anti-Burkholderia gladioli coconut poison variant monoclonal antibody hybridoma cell strain and preparation method thereof

Abstract

The invention discloses an anti-Burkholderia gladioli coconut poison pathogenic variant monoclonal antibody hybridoma cell strain and a preparation method thereof, and relates to the technical field of Burkholderia gladioli. The strain is obtained by immunizing a BALB / c mouse with an antigen prepared from B. gladioli C.; the preparation method comprises the following production steps: S1, preparing an antigen; s2, immunizing the mouse; s3, carrying out cell fusion; s4, selective culture of hybridoma cells; and S5, identifying. Compared with a traditional microculture method and a molecular biological method, the antibody detection system provided by the invention has the advantages that the detection sensitivity is improved, the detection time is effectively shortened, the detection convenience is improved, and a stable basis is provided for an immunoassay method of B. gladioli C.

Description

Technical Field

[0001] This invention relates to the field of Burkholderia gladioli technology, and more specifically to a monoclonal antibody hybridoma cell line against Burkholderia gladioli cocovenenans pathogenic species and its preparation method. Background Technology

[0002] Burkholderia gladioli, formerly known as *Pseudomonas cocovenenans* subsp. *farinofermentans*, is a Gram-negative, aerobic, rod-shaped bacterium belonging to the genus *Burkholderia*. This bacterium poses a significant pathogenicity and food safety risk due to its production of highly toxic bongkrekic acid and toxoflavin. Bongkrekic acid is one of the main toxicants causing foodborne illnesses such as fermented rice noodle poisoning. Its extreme toxicity and heat resistance, coupled with the current lack of an effective antidote, make it a highly significant public health risk. Therefore, it is necessary to establish rapid and effective detection methods for *B. gladioli* to identify and control bongkrekic acid-producing pathogenic bacteria, thereby preventing and reducing the occurrence of foodborne illnesses caused by bongkrekic acid.

[0003] The current methods for detecting *Burkholderia gladioli* C. exhibit significant differences and limitations. According to the National Food Safety Standard for Microbiological Examination of Food: *Burkholderia gladioli* (GB 4789.29-2020), traditional microbial culture methods, which combine biochemical and toxicological tests for determination, offer advantages in terms of intuitive results and comprehensive judgment. However, they require a 15-20 day testing cycle, making them unsuitable for emergency response to food safety incidents. This method relies on multiple verification steps, including colony morphology observation, physiological and biochemical characteristic analysis, and *bongkrekic acid* toxin detection, resulting in a cumbersome procedure and sensitivity to environmental conditions.

[0004] The application of molecular biology techniques has significantly improved detection efficiency and sensitivity, but technical barriers remain. Real-time fluorescence PCR (Huang Jianfei et al., "Establishment and Application of a Rapid Real-Time Fluorescent PCR Detection Method for Burkholderia gladioli") achieves rapid identification of target strains through the design of specific primers and probes, with a detection sensitivity reaching 1×10⁻⁶. 2However, this method requires copies / 5μL and relies on sophisticated instruments and professional operators. Enzymatic recombination isothermal amplification (ERA) based on thermodynamic amplification technology (Lin Zhiwei et al., "Establishment of Three Visualized Rapid Detection Methods for the Pathogenic Species of Burkholderia gladioli based on Thermodynamic Amplification") simplifies amplification conditions and reduces detection time to within 30 minutes, but requires a visual interpretation system or fluorescence detection equipment. Loop-mediated isothermal amplification fluorescence method (Yao Xurong, "Establishment of Loop-mediated Isothermal Amplification Fluorescence Method for Burkholderia gladioli and Burkholderia cocovenenans subsp. oryzae") utilizes fluorescence signals to monitor the amplification process in real time, achieving a detection limit as low as 1.98 pg / μL, but is still limited by instrument cost and operational standardization requirements. While these technologies overcome the time bottleneck of traditional methods, they face practical challenges in grassroots testing scenarios, such as low equipment penetration and high operational barriers.

[0005] Compared to the two methods mentioned above, immunoassay methods offer advantages such as high sensitivity, low cost, and rapid and convenient operation. Enzyme-linked immunosorbent assay (ELISA), as one of the most widely used immunological detection methods, has applications in multiple fields, including food safety testing and medical diagnostics. Constructing an efficient immunological detection method requires, first and foremost, screening for hybridoma cell lines containing monoclonal antibodies against *Burkholderia gladioli* C. Currently, research on monoclonal antibodies against *Burkholderia gladioli* C. is still in its early stages, and the lack of high-purity anti-*Burkholderia gladioli* C. products hinders the establishment of effective specific detection methods.

[0006] Therefore, it is necessary to propose a monoclonal antibody hybridoma cell line against Burkholderia gladioli var. cocovenenans and its preparation method to solve the above problems. Summary of the Invention

[0007] The purpose of this invention is to address the problems mentioned in the background section by providing a monoclonal antibody hybridoma cell line against Burkholderia calendulae var. cocovenenans and its preparation method.

[0008] To achieve the above objectives, the present invention specifically adopts the following technical solution: A hybridoma cell line containing monoclonal antibodies against Burkholderia gladioli cocovenenans pathogenic strain was obtained by immunizing BALB / c mice with antigens prepared from B. gladioli C.; The aforementioned monoclonal antibody hybridoma cell line against Burkholderia gladioli, a pathogenic species of cocovenenans, was prepared by a method comprising the following steps: S1. Preparation of antigens: B. gladioli C. was activated and cultured at an inoculum of 1%, and then B. gladioli C. whole cell antigen, B. gladioli C. fragmentation antigen, and B. gladioli C. outer membrane protein antigen were prepared and stored at 4°C for later use. S2. Immunization of mice: BALB / c mice were immunized with the three antigens B. gladioli and C. obtained in the above steps. S3. Cell fusion: SP2 / 0 mouse myeloma cells were fused with the spleens of immunized BALB / c mice obtained in the above steps and selectively cultured in DMEM medium containing HAT. S4. Selective culture of hybridoma cells: After selectively culturing the cells fused in the above steps in HAT medium, the unfused SP2 / 0 myeloma cells and spleen cells gradually die, and only the fused hybridoma cells survive. S5. Identification: Analyze the culture supernatant of the fused hybridoma cells obtained in the above steps to identify the hybridoma cell line that secretes anti-B. gladioli C. monoclonal antibody as the target cell line.

[0009] As an inventive concept with the same technical solution described above, this invention also claims protection for a method for preparing a hybridoma cell line containing monoclonal antibodies against Burkholderia calceiflora, a species of cocovenenans, for use in preparing the aforementioned hybridoma cell line containing monoclonal antibodies against Burkholderia calceiflora, comprising the following steps: S1. Preparation of antigens: B. gladioli C. was activated and cultured at an inoculum of 1%, and then B. gladioli C. whole cell antigen, B. gladioli C. fragmentation antigen, and B. gladioli C. outer membrane protein antigen were prepared and stored at 4°C for later use. S2. Immunization of mice: BALB / c mice were immunized with the three antigens B. gladioli and C. obtained in the above steps. S3. Cell fusion: SP2 / 0 mouse myeloma cells were fused with the spleens of immunized BALB / c mice obtained in the above steps and selectively cultured in DMEM medium containing HAT. S4. Selective culture of hybridoma cells: After selectively culturing the cells fused in the above steps in HAT medium, the unfused SP2 / 0 myeloma cells and spleen cells gradually die, and only the fused hybridoma cells survive. S5. Identification: Analyze the culture supernatant of the fused hybridoma cells obtained in the above steps to identify the hybridoma cell line that secretes anti-B. gladioli C. monoclonal antibody as the target cell line.

[0010] In step S1, the preparation of antigen, the method for preparing B. gladioli C. whole-cell antigen includes the following steps: S1. Strains culture: Take the B. gladioli C. standard strain, dissolve it in sterile water and then culture it in culture medium. Then expand the culture to the fourth generation and freeze the glycerol bacteria for later use. S2. Inactivation: Take the frozen fourth-generation glycerol bacteria and inoculate them into LB liquid medium at an inoculation rate of 1%. Inactivate them to the logarithmic phase by shaking at 37°C and 200 r / min. Then perform plate counting. After counting, inactivate the bacterial solution with 3‰ formaldehyde solution. Depending on the inactivation situation, the amount of formaldehyde can be increased appropriately.

[0011] Preferably, in step S1, the culture of the strain, the step of culturing using a culture medium is one of the following steps: A: Inoculate with LB liquid medium into a 50 mL Erlenmeyer flask for activation and culture; B: Inoculate onto nutrient agar plates and incubate at 37°C for 24-48 h. Wash off the bacterial growth with sterile water, and then inoculate at a rate of 1% onto LB liquid medium and activate the culture at 37°C and 200 r / min.

[0012] Preferably, in step S1, the preparation of the antigen, the method for preparing the B. gladioli C. fragmented antigen includes the following steps: S1. Activation culture of bacterial strain: Take B. gladioli C. tetragenus glycerol bacteria and inoculate it into LB liquid medium at an inoculation rate of 1% (v / v) for activation culture. Centrifuge the activated bacterial solution at 7000 r / min for 5 min, remove the supernatant, and wash the precipitate with an appropriate amount of sterile PBS buffer. Repeat the washing step to remove residual culture medium components. S2. Protease inhibition treatment: Resuspend the washed bacterial cells in sterile PBS buffer, add 1 mL of PMSF to the resuspended solution, mix thoroughly, so that PMSF in the system can exert its protease inhibitory effect and avoid antigen protein degradation. S3. Ultrasonic disruption: Place the bacterial solution treated above in an ice bath environment and disrupt it using an ultrasonic disruptor. Observe the state of the bacterial solution during the disruption process to ensure that the bacterial cells are fully disrupted. S4. Collection of crushed precipitate: Centrifuge the ultrasonically crushed mixture at 10,000 r / min for 5 min, collect the precipitate, take a small amount of the collected precipitate, and test for viable bacteria by plate coating method. Inactivate the viable bacteria with formalin. S5. Washing and centrifugation: Wash the inactivated precipitate twice with sterile PBS buffer. After each wash, centrifuge to collect the precipitate, remove residual formalin and impurities, weigh the final precipitate, and resuspend it in sterile PBS buffer. The amount of PBS should be 5-10 times the volume of the bacterial cell collection. After resuspending, seal and store at 4°C for later use.

[0013] Preferably, in step S2, the concentration of PMSF is 34 μg / mL.

[0014] Preferably, in step S3, ultrasonic disruption, the ultrasonic disruption power is 200 W for 5 seconds, with a 10-second interval, and the cycle is repeated 25 times.

[0015] Preferably, in step S1, the preparation of the antigen, the method for preparing the B. gladioli C. outer membrane protein antigen includes the following steps: S1. Washing: Activated B. gladioli C was statically cultured at 37°C for 20 h, followed by centrifugation at 7000 r / min for 10 min to collect the bacterial cells. The collected bacterial cells were washed three times with PBS buffer (pH=7.4), and the washed bacterial cells were resuspended in PBS buffer to obtain PBS resuspension. S2. Removal of precipitate: Adjust the pH of the PBS resuspension to acidic using HCl (1 mol / L), and place it on a magnetic stirrer at 4°C for 30 min at 7000 r / min. Then centrifuge the hydrolysate at 4°C for 6 min, discard the precipitate, transfer the supernatant to a new centrifuge tube, and finally centrifuge at 10000 r / min for 30 min. Take the supernatant, remove the residual precipitate, and obtain the supernatant. S3. Filtration: Adjust the pH of the supernatant to alkaline using NaOH solution (1 mol / L), measure the final volume of the liquid, and slowly add ammonium sulfate solid to make the final concentration of the liquid 2.67 mol / L. Let it stand overnight at 4°C to precipitate, then centrifuge at 12000 r / min at 4°C for 6 min. Reconstitute the precipitate with PBS buffer, using 5-10 times the amount of PBS collected from the bacterial cells. Then, put the reconstituted solution into a 10 kDa ultrafiltration tube and ultrafilter at 4000 r / min, continuously adding sterile PBS to completely remove the ammonium sulfate, thus obtaining the prepared outer membrane protein solution. Store at 4°C.

[0016] Preferably, in step S2, removing the precipitate, the pH is adjusted to 2.0 using an HCl solution.

[0017] Preferably, in step S3, filtration, the pH is adjusted to 7.2 using NaOH solution.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Compared with traditional microbial culture methods and molecular biology methods, the antibody detection system provided by this invention improves detection sensitivity and effectively shortens detection time, increasing the convenience of detection and providing a stable foundation for the immunoassay of B. gladioli C.

[0019] 2. This invention significantly enhances antibody immunogenicity by employing a triple immunization strategy combining whole-cell antigen, fragmented antigen, and outer membrane protein antigen. Compared to existing single-antigen immunization techniques, this method can induce BALB / c mice to produce monoclonal antibody responses against different structural layers of the pathogen, providing a highly immunogenic source of spleen cells for subsequent hybridoma cell line screening.

[0020] 3. The antigen preparation process established in this invention has high controllability. By optimizing the ultrasonic disruption parameters and the outer membrane protein extraction conditions, high-purity antigen components can be stably obtained. Attached Figure Description

[0021] Figure 1 This is a growth curve of B. gladioli C. 3 in this invention; Figure 2 This is a growth curve of B. gladioli C. 4 generations in this invention; Figure 3 The whole-cell antigen (2.0 × 10⁻⁶) in this invention 8 ELISA curve of mouse serum antibody titer (CFU); Figure 4 The whole-cell antigen in this invention (1.0 × 10⁻⁶) 8 ELISA curve of mouse serum antibody titer (CFU); Figure 5 The whole-cell antigen in this invention (0.35 × 10⁻⁶) 8 ELISA curve of mouse serum antibody titer (CFU); Figure 6 This is an ELISA curve of the antibody titer in mouse serum from the fragmented antigen group in this invention; Figure 7 This is an ELISA curve showing the serum antibody titer of the outer membrane protein antigen group in mice in this invention. Figure 8 This represents the linear relationship of monoclonal antibodies in this invention; Figure 9 This is the result of the specificity identification of the monoclonal antibody in this invention; Figure 10 This is the technical route for preparing monoclonal antibodies in this invention; Figure 11This is the structure for identifying the monoclonal antibody subtype in this invention. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] This invention immunizes BALB / c mice with three antigens of B. gladioli C., and then uses cell fusion technology, HAT selective medium for culture, and indirect ELISA to screen cell supernatants, ultimately obtaining hybridoma cell lines targeting B. gladioli C. monoclonal antibodies.

[0024] I. Activation of B. gladioli C. Take the freeze-dried B. gladioli C. bacterial powder, add 0.5 mL of sterile water to dissolve it, take 0.2 mL of bacterial solution and culture it in LB liquid medium, culture 0.2 mL in nutrient agar plates, and freeze 0.1 mL to glycerol for later use.

[0025] Step 1: LB liquid culture: Add 0.2 mL of bacterial suspension to 20 mL of LB liquid culture medium and shake at 200 r / min for 18 h to activate. Step 2, Nutrient agar plate: Spread 0.2 mL of bacterial culture onto a plate and incubate for 24-48 h. Wash off the bacterial growth with 0.5 mL of sterile water, and then inoculate 1% of the culture onto LB liquid medium at 200 r / min for expansion culture. Step 3: Monitor the OD600 value of the bacterial culture in a timely manner, and record the growth curves of the 3rd and 4th generations of the bacterial culture to determine the logarithmic growth phase. Also, count the colonies and store the glycerol bacteria for later use.

[0026] Note: Glycerin for sterilization: Mix sterile pure glycerin and bacterial solution in a cryovial at a ratio of 3:7 and freeze at -80°C.

[0027] Reference for the third generation growth curves of B. gladioli C. Figure 1 Reference for the 4th generation growth curves of B. gladioli C. Figure 2 .

[0028] This invention uses activated B. gladioli C. 4th generation glycerol bacteria to prepare whole bacterial antigens for immunizing BALB / c mice.

[0029] II. B. gladioli C. Preparation of three antigens The above-prepared B. gladioli C. tetragenus glycerol bacteria were used to prepare three antigens.

[0030] 1. The specific method for preparing whole-cell antigen is as follows: Step 1: Bacterial activation: Take the frozen glycerol bacteria out of the -80℃ freezer and inoculate them into LB liquid medium at a 1% inoculum. Activate the bacteria to the logarithmic growth phase at 37℃ and 200 r / min. Perform plate counting on the activated bacterial solution. Step 2, Bacterial Inactivation: After counting the bacteria using the plate plating method, add 3‰ (v / v) formaldehyde solution (commercially available formalin concentration calculated as 100%) for inactivation at room temperature for more than 12 hours, shaking several times during the process. Inactivation Verification: After plating the inactivated bacterial solution and finding no viable bacteria, centrifuge at 7000 r / min for 5 min to remove the culture medium, retaining the bacterial cell collection. Step 3: Gently resuspend the bacterial cell collection in PBS (5-10 times the volume of the bacterial cell collection), centrifuge at 7000 r / min for 5 min, collect the bacterial cells, and repeat twice to wash away macromolecular pyrogens in the culture medium. Finally, aliquot the bacterial cells into sterile centrifuge tubes and store at 4℃ for later use.

[0031] 2. The specific method for preparing fragmented antigen is as follows: Step 1: Take the frozen fourth-generation glycerol bacteria and inoculate them into LB liquid medium at a 1% inoculum. Activate to the logarithmic growth phase at 37°C and 200 r / min. Centrifuge at 7000 r / min for 5 min to remove the medium and retain the bacterial cell collection. Wash the bacterial cell collection twice with PBS to remove large molecular pyrogens from the medium. Step 2: Resuspend the cells in a small amount of PBS (depending on the loading volume of the sonication cell disruptor), then add 1 mL (34 μg / mL) of PMSF to inactivate the protease. In an ice bath, sonicate at 200 W for 5 seconds, with 10-second intervals, repeating 25 times. If the bacterial culture does not become clear, increase the number of cycles. Collect the precipitate from the disrupted solution at 10000 r / min for 5 minutes. Step 3: Take a small amount of the above precipitate, spread it on a plate to check for live bacteria. If live bacteria are present, inactivate the precipitate with formalin, wash it twice with PBS, centrifuge and collect it, weigh the precipitate, resuspend it in sterile PBS (the amount of PBS should be 5-10 times the volume of the collected bacteria), and store it at 4°C for later use.

[0032] 3. The specific preparation method for outer membrane protein antigen is as follows: Step 1: Incubate the activated *B. gladioli* C. at 37°C for 20 h, then centrifuge at 7000 r / min for 10 min to collect the bacterial cells. Wash the collected bacterial cells three times with PBS buffer (pH=7.4) to remove residual LB medium. Resuspend the washed bacterial cells in 6 mL of PBS buffer. Step 2: Adjust the pH of the resuspension to 2.0 with HCl (1 mol / L) and stir on a magnetic stirrer at 4°C for 30 min for acid hydrolysis. Centrifuge the hydrolysate at 7000 r / min and 4°C for 6 min, discard the precipitate, and transfer the supernatant to a new centrifuge tube. Centrifuge again at 10000 r / min for 30 min, collect the supernatant, and remove any remaining precipitate. Adjust the pH of the supernatant to 7.2 with NaOH solution (1 mol / L). Step 3: Measure the final volume of the liquid, slowly add ammonium sulfate solid to achieve a final concentration of 2.67 mol / L, and let it stand overnight at 4°C to precipitate; centrifuge at 12000 r / min, 4°C for 6 min, and reconstitute the precipitate with PBS buffer (the amount of PBS used is 5-10 times that of the bacterial cell collection), then put the reconstituted solution into a 10 kDa ultrafiltration tube, ultrafilter at 4000 r / min, and continuously add sterile PBS to completely remove the ammonium sulfate; Step 4: Store the prepared outer membrane protein solution at 4°C for later use.

[0033] III. Immunity in Mice After the three antigens of B. gladioli and C. were emulsified with an equal volume of Freund's complete adjuvant, an injectable solution was obtained and BALB / c mice were immunized by subcutaneous injection at multiple points on the back.

[0034] Step 1, First Immunization: Mix Freund's complete adjuvant and B. gladioli C. antigen in equal volumes and emulsify using a vortex mixer until it is milky white and shows no obvious diffusion in water. Immunize each mouse with a 200 μL injection volume (adjuvant to immunogen 1:1) and administer the immunization at multiple subcutaneous sites on the back. Step 2, two to five immunizations: Three weeks after the first immunization, equal volumes of Freund's incomplete adjuvant and B. gladioli C. antigen were mixed and thoroughly emulsified using a vortex mixer. The mixture was then injected subcutaneously at multiple points on the back of the mouse for immunization. Seven days after each immunization, blood was collected by tail amputation using ophthalmic scissors to determine serum titers. Step 3, booster immunization: If the titer does not meet the requirements for cell fusion, a sixth immunization must be performed using the above method. Three days before cell fusion, mix the antigen with an equal volume of sterile PBS, without adding adjuvants, and inject one mouse with the target titer into the peritoneum for booster immunization to induce cell fusion.

[0035] Reference image for experimental results of hexaimmune titer of whole bacterial antigen group Figures 3 to 7 .

[0036] After screening and comparison, the whole bacterial antigen group (2.0 × 10⁻⁶) was selected. 8 CFU-1 mice were used as the cell fusion subjects, and the dose of the initial immunization was twice that of the booster immunization, i.e., 4.0 × 10⁻⁶ mice were injected intraperitoneally. 8 CFU / 200 μL / only unadjuvanted B. gladioli C. dilution.

[0037] IV. Cell Fusion Three days after the initial immunization, cell fusion was performed using the standard PEG method, with the following specific steps: Step 1: Preparation of SP2 / 0 myeloma cells: Cell fusion requires SP2 / 0 cells to be round and translucent. One week prior to fusion, SP2 / 0 mouse myeloma cells were resuscitated and cultured in a 5% CO2 incubator. Cell status was observed daily to ensure good cell condition at fusion. Approximately two flasks (T75 cm⁻¹) are needed for cell fusion. 2 SP2 / 0 cells (cell flasks) grown to 80%-90% were collected. Upon fusion, the culture medium was discarded, and the cells were washed twice with sterile PBS. Then, the cells were blown off the flask wall with DMEM culture medium and collected into a 50 mL centrifuge tube. The volume was increased to 20 mL, and the cells were centrifuged (8000 rpm, 5 min). After processing, the cells were resuspended in 20 mL of DMEM culture medium. Step 2: Remove the mouse eyeballs, collect blood, incubate at 37℃ for 2 h, centrifuge (6000 rpm, 10 min), aliquot and store at -20℃ as a positive control for screening. Immerse the mouse in 75% alcohol for 3-5 min, fix the mouse on a work surface, and aseptically remove the mouse spleen. Place the spleen on a small beaker with a 300-mesh sieve, gently cut away the connective tissue around the spleen with scissors, add DMEM medium and grind into the beaker, then transfer to a 50 mL centrifuge tube, add DMEM medium to 40 mL, centrifuge (1500 rpm, 5 min), and resuspend in 20 mL DMEM medium. Mix the resuspended SP2 / 0 cells with the spleen cells, centrifuge (1500 rpm, 5 min), discard the supernatant, gently tap the cell clump at the bottom of the centrifuge tube to disperse the cells, and place in a 37℃ water bath beaker. Step 3: For cell fusion, prepare 1 mL of preheated PEG 1500 (37℃), double-distilled water, and DMEM medium in advance. Add 1 mL of preheated PEG 1500 (37℃) slowly at first, then quickly over 1 minute. Let stand for 90 seconds. Then, slowly add 15 mL of DMEM medium in three stages to terminate the fusion reaction: 1 mL / 30 s, 3 mL / 30 s, and 11 mL / 30 s. Incubate at 37℃ for 5 minutes. Add DMEM medium to a final volume of 40 mL. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and gently tap the cell clumps at the bottom of the centrifuge tube. Add preheated HAT medium and gently pipette to mix the cell clumps. Add 200 μL per well to a 96-well cell culture plate and incubate at 37℃ in a 5% CO2 incubator. Observe cell growth on the second day. After 7-10 days, examine the cell supernatant and select positive wells.

[0038] The results of ELISA screening of positive wells in polyclonal cell supernatant are shown in Tables 1 to 10 below.

[0039] Table 1

[0040] Table 2

[0041] Table 3

[0042] Table 4

[0043] Table 5

[0044] Table 6

[0045] Table 7

[0046] Table 8

[0047] Table 9

[0048] Table 10

[0049] V. Selection and cryopreservation of hybridoma cell lines Step 1: When the cells grow to 1 / 3 of the bottom area, perform ELISA screening. One day in advance, completely change the medium with HT medium. On the second day, perform indirect ELISA screening. Transfer the strongly positive wells to a 24-well cell culture plate for expansion culture. When the cells grow to 1 / 2 of the bottom area of ​​the well, use indirect ELISA to detect the cell supernatant again. Step 2, cryopreservation of hybridoma cells: Take the positive polyclonal cell line from the supernatant for cryopreservation. When the cells in the 24-well plate cover 70%-80% of the bottom area, use a 1mL pipette to pipette the cells off and centrifuge them in a centrifuge tube at 1000 rpm for 10 min. Discard the supernatant and resuspend the cell pellet in 1 mL of cell cryopreservation solution. Store the cells in a freezer at -80℃. Step 3: Based on the positive control wells, five monoclonal antibodies, namely 1E2, 2E3, 3E12, 3G10, and 3H8, were screened out and cryopreserved when the cells reached a bottom area of ​​70%-80%.

[0050] Refer to Tables 11 and 12 for screening positive wells of subclonal cell supernatant.

[0051] Table 11

[0052] Table 12

[0053] Reference Table 13 for screening positive wells of monoclonal cell supernatant.

[0054] Table 13 Screening of positive wells from monoclonal cell supernatant

[0055] VI. Identification of hybridoma cells secreting anti-B. gladioli C. monoclonal antibodies 1. Take the supernatant from the 24-well cell culture plate and perform the following steps: Step 1, Antigen Coating: Determine the optimal coating concentration using the checkerboard method, setting the concentration to 1.0 × 10⁻⁶. 8 Inactivated B. gladioli C. culture (by plate counting) with CFU was added to each well of a 96-well plate and incubated overnight at 4°C or for 2 h at 37°C. Step 2, washing the plate: Pour out the liquid in the microplate that has been left overnight or at 37°C, shake off the excess water, wash the plate with the prepared PBST, fill each well, shake off the excess water, and repeat 3 times. Step 3, Blocking: 5% skim milk, 300 μL / well, block, 37℃, 1 h, wash 3 times with PBST; Step 4: Add the sample to be tested: Add 100 μL of the sample to be tested to the coated wells, perform replicates for each dilution, and perform a blank control at the same time. Incubate at 37℃ for 1 h, and wash the plate 3 times. Step 5: Incubate with enzyme-labeled secondary antibody: Add goat anti-mouse IgG-HRP, 100 μL / well, incubate at 37℃ for 0.5 h, and wash the plate 5 times; Step 6, color development and termination: ① Add 100 μL of chromogenic substrate and develop color in the dark for 15 min; ② 50 μL / well stop solution and detect the OD value at 450 nm using a microplate reader within 5 min.

[0056] The solution was prepared as follows in the previous step: Phosphate buffer (0.01M PBS): 8.0 g NaCl, 0.2 g KCl, 0.24 g KH2PO4, 1.44 g Na2HPO4, dissolved in 800 mL distilled water, pH adjusted to 7.2-7.4 with NaOH or HCl, and brought to a final volume of 1000 mL. 0.1% PBST washing solution: Dissolve 8.0 g NaCl, 0.2 g KCl, 0.24 g KH2PO4, and 1.44 g Na2HPO4 in 800 mL of distilled water. Adjust the pH to 7.2-7.4 with NaOH or HCl, bring the volume to 1000 mL, and add 1 mL of Tween-20. Blocking solution: 5 g skim milk dissolved in 100 mL PBST; TMB substrate developing solution: Mix equal volumes of solution A and solution B; Termination solution (2M H2SO) 4） Mix 178.3 mL of distilled water with 21.7 mL of 98% concentrated sulfuric acid.

[0057] 2. Sensitivity determination The antigen is B. gladioli C. Let 10. 6 10 5 10 4 10 3 10 2 10 1 Six concentration gradients of CFU / mL.

[0058] Using antibodies diluted to approximately 1.0 OD450, the remaining steps are the same as for titer determination. The sensitivity of the antibody detection is determined by measuring the number of target bacteria that can be detected by the whole-cell antibody at its optimal working dilution.

[0059] Adoption of Monoclonal Antibody Sensitivity Identification Results (Table 14): Table 14. Results of Monoclonal Antibody Sensitivity Identification

[0060] Figure 8 This is a schematic diagram illustrating the linear relationship of monoclonal antibodies.

[0061] 3. Specificity identification Common foodborne pathogens, such as *Escherichia coli*, *Listeria monocytogenes*, *Salmonella typhimurium*, and *Staphylococcus aureus*, were selected as control strains for specificity testing. These five strains were coated onto 96-well plates, and the optimal dilution of each antibody was used for the experiment. Antibody specificity was determined by a ratio of OD450nm of the test well to OD450nm of the blank well, which was ≥2.1.

[0062] Figure 9 The image shows the results of the specific identification of monoclonal antibodies.

[0063] Figure 10 This is a technical roadmap for monoclonal antibody preparation.

[0064] Figure 11 This is a schematic diagram of the structure for identifying monoclonal antibody subtypes.

[0065] This invention successfully constructed a hybridoma cell line containing monoclonal antibodies against Burkholderia gladioli C. and its preparation method. A triple antigen immunization strategy (whole-cell antigen, fragmentation antigen, and outer membrane protein antigen) significantly enhanced the immunogenicity of the antibody. Experimental results showed that the prepared hybridoma cell line could stably secrete high-titer, high-specificity monoclonal antibodies, and five positive cell lines (1E2, 2E3, 3E12, 3G10, and 3H8) were identified.

[0066] Compared with traditional microbial culture methods (15-20 days) and molecular biology techniques, the immunoassay system established in this invention has the following advantages: 1. High sensitivity and wide linear range: The linear relationship is y = 0.1946x - 0.1988, R 2 =0.9726; IC 50 =3.89x10 3 cfu / mL; linear range was 1.12 x 10⁻⁶. 2 -1.35 x10 5 cfu / mL; 2. Fast and convenient: Reduces testing time to a few hours, requiring no complex instruments; 3. High specificity: No cross-reactivity with common foodborne pathogens such as Escherichia coli and Listeria monocytogenes; 4. Good stability: Hybridoma cell lines can stably secrete antibodies.

[0067] This invention provides a key immunological tool for the rapid detection of B. gladioli C., filling the gap in the lack of specific antibodies in this field, and providing important technical support for food safety risk monitoring and emergency response to public health emergencies.

Claims

1. A hybridoma cell line containing monoclonal antibodies against Burkholderia gladioli var. cocovenenans pathogenic strains, characterized in that, This strain was obtained by immunizing BALB / c mice with an antigen prepared from B. gladioli C.; The aforementioned monoclonal antibody hybridoma cell line against Burkholderia gladioli, a pathogenic species of cocovenenans, was prepared by a method comprising the following steps: S1. Preparation of antigens: B. gladioli C. was activated and cultured at an inoculum of 1%, and then B. gladioli C. whole cell antigen, B. gladioli C. fragmentation antigen, and B. gladioli C. outer membrane protein antigen were prepared and stored at 4°C for later use. S2. Immunization of mice: BALB / c mice were immunized with the three antigens B. gladioli and C. obtained in the above steps. S3. Cell fusion: SP2 / 0 mouse myeloma cells were fused with the spleens of immunized BALB / c mice obtained in the above steps and selectively cultured in DMEM medium containing HAT. S4. Selective culture of hybridoma cells: After selectively culturing the cells fused in the above steps in HAT medium, the unfused SP2 / 0 myeloma cells and spleen cells gradually die, and only the fused hybridoma cells survive. S5. Identification: Analyze the culture supernatant of the fused hybridoma cells obtained in the above steps to identify the hybridoma cell line that secretes anti-B. gladioli C. monoclonal antibody as the target cell line.

2. A method for preparing a hybridoma cell line containing monoclonal antibodies against *Burkholderia gladioli* cocovenenans pathogenic strain, used to prepare the hybridoma cell line containing monoclonal antibodies against *Burkholderia gladioli* cocovenenans pathogenic strain as described in claim 1, comprising the following steps: S1. Preparation of antigens: B. gladioli C. was activated and cultured at an inoculum of 1%, and then B. gladioli C. whole cell antigen, B. gladioli C. fragmentation antigen, and B. gladioli C. outer membrane protein antigen were prepared and stored at 4°C for later use. S2. Immunization of mice: BALB / c mice were immunized with the three antigens B. gladioli and C. obtained in the above steps. S3. Cell fusion: SP2 / 0 mouse myeloma cells were fused with the spleens of immunized BALB / c mice obtained in the above steps and selectively cultured in DMEM medium containing HAT. S4. Selective culture of hybridoma cells: After selectively culturing the cells fused in the above steps in HAT medium, the unfused SP2 / 0 myeloma cells and spleen cells gradually die, and only the fused hybridoma cells survive. S5. Identification: Analyze the culture supernatant of the fused hybridoma cells obtained in the above steps to identify the hybridoma cell line that secretes anti-B. gladioli C. monoclonal antibody as the target cell line.

3. The method for preparing a hybridoma cell line with monoclonal antibodies against Burkholderia gladioli var. cocovenenans pathogenicity according to claim 2, characterized in that, In step S1, the preparation of the antigen, the method for preparing the whole bacterial antigen of B. gladioli C. includes the following steps: S1. Strains culture: Take the B. gladioli C. standard strain, dissolve it in sterile water and then culture it in culture medium. Then expand the culture to the fourth generation and freeze the glycerol bacteria for later use. S2. Inactivation: Take the frozen fourth-generation glycerol bacteria and inoculate them into LB liquid medium at an inoculation rate of 1%. Inactivate them to the logarithmic phase by shaking at 37°C and 200 r / min. Then perform plate counting. After counting, inactivate the bacterial solution with 3‰ formaldehyde solution. Depending on the inactivation situation, the amount of formaldehyde can be increased appropriately.

4. The method for preparing a hybridoma cell line with monoclonal antibodies against Burkholderia gladioli var. cocovenenans pathogenicity according to claim 3, characterized in that, In step S1, the culture of the strain, the step of using a culture medium for cultivation is one of the following steps: A: Inoculate with LB liquid medium into a 50 mL Erlenmeyer flask for activation and culture; B: Inoculate onto nutrient agar plates, incubate at 37°C for 24-48 h, wash the bacterial growth with sterile water, and then inoculate at a rate of 1% onto LB liquid medium at 37°C and 200 r / min for activation culture.

5. The method for preparing a hybridoma cell line with monoclonal antibodies against Burkholderia gladioli var. cocovenenans pathogenicity according to claim 2, characterized in that, In step S1, the preparation of the antigen, the method for preparing the fragmented antigen of B. gladioli C. includes the following steps: S1. Activation culture of bacterial strain: Take B. gladioli C. tetragenus glycerol bacteria and inoculate it into LB liquid medium at an inoculation rate of 1% (v / v) for activation culture. Centrifuge the activated bacterial solution at 7000 r / min for 5 min, remove the supernatant, and wash the precipitate with an appropriate amount of sterile PBS buffer. Repeat the washing step to remove residual culture medium components. S2. Protease inhibition treatment: Resuspend the washed bacterial cells in sterile PBS buffer, add 1 mL of PMSF to the resuspended solution, mix thoroughly, so that PMSF in the system can exert its protease inhibitory effect and avoid antigen protein degradation. S3. Ultrasonic disruption: Place the bacterial solution treated above in an ice bath environment and disrupt it using an ultrasonic disruptor. Observe the state of the bacterial solution during the disruption process to ensure that the bacterial cells are fully disrupted. S4. Collection of crushed precipitate: Centrifuge the ultrasonically crushed mixture at 10000 r / min for 5 min, collect the precipitate, take a small amount of the collected precipitate, and test for viable bacteria by plate coating method. Inactivate the viable bacteria with formalin. S5. Washing and centrifugation: Wash the inactivated precipitate twice with sterile PBS buffer. After each wash, centrifuge to collect the precipitate, remove residual formalin and impurities, weigh the final precipitate, and resuspend it in sterile PBS buffer. The amount of PBS should be 5-10 times the volume of the bacterial cell collection. After resuspending, seal and store at 4°C for later use.

6. The method for preparing a hybridoma cell line with monoclonal antibodies against Burkholderia gladioli var. cocovenenans pathogenicity according to claim 5, characterized in that, In step S2, the protease inhibition treatment, the concentration of PMSF is 34 μg / mL.

7. The method for preparing a hybridoma cell line with monoclonal antibodies against Burkholderia gladioli var. cocovenenans pathogenicity according to claim 5, characterized in that, In step S3, ultrasonic disruption, the ultrasonic disruption power is 200 W for 5 seconds, with a 10-second interval, and the cycle is repeated 25 times.

8. The method for preparing a hybridoma cell line with monoclonal antibodies against Burkholderia gladioli var. cocovenenans pathogenicity according to claim 2, characterized in that, In step S1, the preparation of the antigen, the method for preparing the B. gladioli C. outer membrane protein antigen includes the following steps: S1. Washing: Activated B. gladioli C was statically cultured at 37°C for 20 h, followed by centrifugation at 7000 r / min for 10 min to collect the bacterial cells. The collected bacterial cells were washed three times with PBS buffer (pH=7.4), and the washed bacterial cells were resuspended in PBS buffer to obtain PBS resuspension. S2. Removal of precipitate: Adjust the pH of the PBS resuspension to acidic using HCl (1 mol / L), and place it on a magnetic stirrer at 4°C for 30 min at 7000 r / min. Then centrifuge the hydrolysate at 4°C for 6 min, discard the precipitate, transfer the supernatant to a new centrifuge tube, and finally centrifuge at 10000 r / min for 30 min. Take the supernatant, remove the residual precipitate, and obtain the supernatant. S3. Filtration: Adjust the pH of the supernatant to alkaline using NaOH solution (1 mol / L), measure the final volume of the liquid, and slowly add ammonium sulfate solid to make the final concentration of the liquid 2.67 mol / L. Let it stand overnight at 4°C to precipitate, then centrifuge at 12000 r / min at 4°C for 6 min. Reconstitute the precipitate with PBS buffer, using 5-10 times the amount of PBS collected from the bacterial cells. Then, put the reconstituted solution into a 10 kDa ultrafiltration tube and ultrafilter at 4000 r / min, continuously adding sterile PBS to completely remove the ammonium sulfate, thus obtaining the prepared outer membrane protein solution. Store at 4°C.

9. The method for preparing a hybridoma cell line with monoclonal antibodies against Burkholderia gladioli var. cocovenenans pathogenicity according to claim 8, characterized in that, In step S2, the pH is adjusted to 2.0 using HCl solution to remove the precipitate.

10. The method for preparing a hybridoma cell line with monoclonal antibodies against Burkholderia gladioli var. cocovenenans pathogenicity according to claim 8, characterized in that, In step S3, during filtration, the pH is adjusted to 7.2 using NaOH solution.

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