Hybridoma cell strain capable of secreting tildipirosin monoclonal antibody and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain capable of secreting a tildipirosin monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of immunodetection. The monoclonal antibody secreted by the hybridoma cell strain provided by the invention has good sensitivity and specificity to the tildipirosin, wherein the IC50 value to the tildipirosin is 20 ng / mL. The monoclonal antibody provided by the invention has no cross reaction on structural analogues of tildipirosin, such as erythromycin, streptomycin, azithromycin and spiramycin, and has good specificity, so that low-concentration tildipirosin can be accurately detected.

Description

Technical Field

[0001] This invention relates to the field of immunoassay, and in particular to a hybridoma cell line that secretes a monoclonal antibody against thiazolinone and its applications. Background Technology

[0002] Tildipirosin (TDS) is a novel, animal-specific, sixteen-membered ring macrolide semi-synthetic antibiotic, a derivative of tylosin. Tildipirosin exerts its potent antibacterial activity primarily by inhibiting bacterial protein synthesis. Due to its long half-life, high bioavailability, and broad antibacterial spectrum, it is widely used to treat and prevent respiratory diseases in pigs and cattle caused by *Actinomyces pleuropneumoniae*, *Pasteurella multocida*, and other bacteria. However, tildipirosin is slowly metabolized in animals, potentially leading to drug residues in edible tissues such as muscle and liver. Long-term consumption of animal-derived foods containing tildipirosin residues may pose biosafety risks, such as causing intestinal flora imbalance, the development of drug-resistant strains, or potential poisoning and allergic reactions in humans.

[0003] Currently, to protect consumers from the harm of drug residues, many countries have stipulated maximum limits for taliroxin in different tissues. In the European Union, the maximum permitted level of taliroxin in pork is 1200 µg / kg; the maximum permitted level in beef and lamb is 400 µg / kg; and the content of taliroxin in food must be strictly controlled. Therefore, research on methods for detecting taliroxin is of great significance.

[0004] Currently, there are few analytical methods for determining teldrone content, mainly including instrumental methods such as gas chromatography-tandem mass spectrometry (GC-MS / MS), high-performance liquid chromatography-UV, and liquid chromatography-tandem mass spectrometry (LC-MS / MS). These methods are complex to operate and require cumbersome sample processing, which is not conducive to the detection of large batches of samples in actual production. Therefore, there is an urgent need to establish a detection method that is simple to operate, highly sensitive, and highly specific. Enzyme-linked immunosorbent assay (ELISA) is a highly efficient, sensitive, and rapid detection method, and it is increasingly being used in food safety testing. The prerequisite for using ELISA to detect teldrone is to obtain a monoclonal antibody that can accurately detect teldrone. Therefore, finding a monoclonal antibody that can accurately detect teldrone is crucial. However, existing monoclonal antibodies cannot simultaneously achieve high sensitivity and high specificity for teldrone. Therefore, there is an urgent need to find a new monoclonal antibody to achieve efficient detection of teldrone. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a monoclonal antibody in the prior art that can accurately detect low concentrations of tepidilogen.

[0006] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes a monoclonal antibody against teldirocin and its application. The monoclonal antibody secreted by the hybridoma cell line of this invention exhibits good sensitivity and specificity to teldirocin, with an IC50 value of [missing information - likely an IC50 value]. 50 The value is 20 ng / mL. Furthermore, the monoclonal antibody of this invention shows no cross-reactivity with structural analogs of tepidiloxine, such as erythromycin, streptomycin, azithromycin, and spiramycin, exhibiting good specificity. Therefore, it can accurately detect low concentrations of tepidiloxine.

[0007] The first objective of this invention is to provide a hybridoma cell line, characterized in that the hybridoma cell line is named a monoclonal cell line and has the accession number CGMCC No. 46522.

[0008] Furthermore, the hybridoma cell line is obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structural formula of the hapten is shown in Formula I:

[0009] .

[0010] Furthermore, the hapten of the present invention is obtained by oxime reaction of temeroxanil technical and CMO. The preparation method of the hapten of the present invention is simple and the product yield is high.

[0011] Furthermore, the complete antigen is obtained from the hapten coupled with a carrier protein.

[0012] Furthermore, the carrier protein includes bovine serum albumin.

[0013] A second objective of this invention is to provide an application of the described hybridoma cell line in the detection of teldrone.

[0014] A third objective of this invention is to provide a monoclonal antibody secreted by the hybridoma cell line described above.

[0015] The fourth objective of this invention is to provide an application of the above-mentioned monoclonal antibody in the detection of teldrone.

[0016] A fifth objective of this invention is to provide a novel diagnostic product for tadalafil, the diagnostic product comprising the aforementioned monoclonal antibody.

[0017] Furthermore, the test product also includes a coating agent.

[0018] Furthermore, the coating is prepared from a hapten-conjugated carrier protein, wherein the carrier protein includes chicken ovalbumin.

[0019] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0020] The monoclonal antibody secreted by the hybridoma cell line of the present invention exhibits good sensitivity and specificity to teldextrin, wherein the IC50 value for teldextrin is [not specified]. 50 The value is 20 ng / mL. Furthermore, the monoclonal antibody of this invention shows no cross-reactivity with structural analogs of tepidiloxine, such as erythromycin, streptomycin, azithromycin, and spiramycin, exhibiting good specificity. Therefore, it can accurately detect low concentrations of tepidiloxine.

[0021] Preservation of biological materials

[0022] The monoclonal cell line NPEO was deposited on April 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46522, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0023] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0024] Figure 1 This is a synthetic route diagram of the thiamethoxam hapten of the present invention;

[0025] Figure 2 This is the standard curve of the inhibition of teldrolidine monoclonal antibody against teldrolidine in this invention. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0028] (1) The culture media involved in the following examples are as follows:

[0029] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cysteine ​​dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5. L-Tyrosine 23.19, L-Valine 20, Para-aminobenzoic acid 1, Calcium nitrate 100, Anhydrous magnesium sulfate 48.84, Anhydrous sodium dihydrogen phosphate 676.13, Potassium chloride 400, Sodium chloride 6000, Glucose 2000, Reduced glutathione 1, Phenol red 5, L-Glutamine 300, Biotin 0.2, D-Calcium pantothenate 0.25, Folic acid 1, I-Inositol 35, Nicotinamide 1, Choline chloride 3, Pyridoxine hydrochloride 1, Riboflavin 0.2, Thiamine hydrochloride 1, Vitamin B12 0.005, Sodium bicarbonate 2000.

[0030] (2) The reagents involved in the following examples are as follows:

[0031] Carbonate buffer (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800 mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000 mL. Store at 4°C for later use.

[0032] Phosphate buffer (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, dissolved in 800 mL pure water, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000 mL with ultrapure water.

[0033] Washing buffer (PBST): Add 0.5 mL of Tween-20 to 1000 mL of 0.01 mol / L pH 7.4 PBS solution;

[0034] PBST: PBS containing 0.05% Tween-20;

[0035] Antibody diluent: a washing solution containing 0.1% gelatin;

[0036] TMB colorimetric solution: Solution A: 18.43 g Na2HPO4·12H2O, 9.33 g citric acid, diluted to 1000 mL with pure water; Solution B: 60 mg TMB dissolved in 100 mL ethylene glycol. Mix solutions A and B in a volume ratio of 5:1 to obtain the TMB colorimetric solution. Mix fresh before use.

[0037] (3) The detection methods involved in the following embodiments are as follows:

[0038] The method for detecting the inhibition rate of teldrone sulfamethoxazole (TDS) is as follows: The optimal antigen and antibody concentrations for icELISA are selected using a checkerboard assay. The antigen is diluted to 1, 0.3, and 0.1 μg / mL with carbonate buffer (CBS), and the antibody is diluted to 0.3, 0.1, 0.03, and 0.01 μg / mL with antibody diluent. After selecting the optimal operating point, TDS standard is diluted to concentrations of 20, 60, and 120 ng / mL. Following the icELISA procedure, the inhibition curves of the TDS standard are obtained using Origin 2024, and the IC50 is calculated. 50 .

[0039] Example 1: Synthesis of tadalafil hapten

[0040] The hapten tadalafil is obtained by reacting the technical grade tadalafil with a CMO to yield a product containing a carboxyl group. The synthetic route is as follows: Figure 1 As shown. The specific reaction steps are as follows:

[0041] Reaction A: 50 mg (0.51 mmol) of tebuconazole and 150 mg (1.2 mmol) of CMO were dissolved in pyridine. After being protected from light, the solution was reacted at 70 °C for 4 h using a magnetic stirrer. After the reaction was complete, the solution was dried under a nitrogen stream and extracted three times with 1 mL of ultrapure water and an equal volume of chloroform. The lower organic phases were combined and dried under a nitrogen stream to obtain the reaction product TDS-CMO.

[0042] The crude sample was purified by recrystallization. The crude sample was dissolved in methanol. After complete dissolution, it was allowed to stand overnight at room temperature, resulting in the precipitation of a brown precipitate. This precipitate was filtered through filter paper, washed 2-3 times with petroleum ether, and dried at 50°C to obtain a brown precipitate (37.5 mg), which was the hapten TDS, with a yield of 75.0%.

[0043] Example 2: Preparation of complete antigen

[0044] The hapten TDS prepared in Example 1 was conjugated with bovine serum albumin (BSA) using the carbodiimide method to obtain the complete antigen TDS-EDC-BSA. The specific steps are as follows:

[0045] Weigh 3.55 mg of the hapten TDS, 1.52 mg of N-hydroxysuccinimide, and 2.53 mg of 1-ethylcarbodiimide hydrochloride prepared in Example 1, dissolve them in 300 μL of N,N-dimethylformamide (referred to as solution A), and stir at room temperature for 4-6 h. Weigh 6.0 mg of BSA (TDS to BSA molar ratio of 20:1), add 2 mL of carbonate buffer solution (the dissolved BSA protein is called solution B), and add solution A dropwise to solution B at room temperature. Adjust the pH of the mixture to 8-9 with 1 M NaOH solution and react overnight at room temperature to obtain the conjugate TDS-EDC-BSA. Cut an 8 cm dialysis bag, boil it in boiling water for 3 min and cool it, then store it in deionized water at 4℃ for later use. Place the conjugate TDS-EDC-BSA in a dialysis bag and dialyze it in 0.01 mol / L PBS, changing the solution every 8 h. Dialyze for 3 days to obtain the complete antigen TDS-EDC-BSA, which should be removed and stored at -20℃.

[0046] Example 3: Preparation of the coating agent

[0047] The hapten TDS prepared in Example 1 was conjugated with chicken oocyte albumin OVA using the carbodiimide method to obtain coated TDS-EDC-OVA. The specific steps are as follows:

[0048] Weigh out 3.55 mg of the hapten TDS, 1.96 mg of N-hydroxysuccinimide, and 2.53 mg of 1-ethylcarbodiimide hydrochloride prepared in Example 1, dissolve them in 50 μL of N,N-dimethylformamide (referred to as solution A), and stir at room temperature for 4-6 h. Weigh 3 mg of OVA (TDS to OVA molar ratio of 20:1), add 2 mL of carbonate buffer solution (OVA protein dissolved, referred to as solution B), and add solution A dropwise to solution B at room temperature. Adjust the pH of the mixture to 8-9 with 1 M NaOH solution and react overnight at room temperature to obtain the conjugate TDS-EDC-OVA. Cut an 8 cm dialysis bag, boil it in boiling water for 3 min and cool it, then store it in deionized water at 4℃ for later use. Place the conjugate TDS-EDC-OVA in a dialysis bag and dialyze it in 0.01 mol / L PBS, changing the solution every 8 h. Dialyze for 3 days to obtain the complete antigen TDS-EDC-OVA, remove it and store it at -20℃.

[0049] Example 4: Preparation of hybridoma cell lines

[0050] 1. Immunization of mice: Healthy 6-8 week old BALB / c mice were selected for immunization. For the first immunization, 100 μg of tepidiloxin complete antigen was emulsified with an equal volume of complete Freund's adjuvant and injected subcutaneously at multiple sites on the neck and back of the mice. Four weeks later, a booster immunization was performed with half the dose of complete antigen (50 μg / mouse) emulsified with incomplete Freund's adjuvant. Subsequent booster immunizations were given at 3-week intervals. For the final sprint immunization, the dose was again halved (25 μg / mouse), and the complete antigen was diluted with physiological saline and injected intraperitoneally. After the third immunization, tail-disconnected blood samples were collected for testing. The serum titer and IC50 were detected using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). 50 Choose high-performance ICs 50 Low-grade mice were fused;

[0051] 2. Cell fusion and screening

[0052] (1) Three days after the sprint immunization, cell fusion was performed according to the conventional PEG 4000 (polyethylene glycol) method. The specific steps are as follows:

[0053] a. Collection of SP2 / 0 tumor cells: 7-10 days before fusion, culture SP2 / 0 tumor cells in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The required number of SP2 / 0 tumor cells before fusion should reach 1-4 × 10⁻⁶ cells / year. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. During fusion, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counting is performed.

[0054] b. After euthanizing the mice by cervical dislocation, immediately sterilize them in 75% alcohol for about 5 minutes. Aseptically remove the spleen, gently grind it with a syringe tip, and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect 50 mL of the suspension in a sterile centrifuge tube, centrifuge at 1200 r / min for 8 minutes, wash the spleen cells with RPMI-1640 medium, remove any large tissue impurities, and repeat the process three times. After the final centrifugation, dilute the spleen cells to a specific volume, count them, and set aside for later use.

[0055] c. Fusion process (7 min): At min 1, add 1 mL of PEG 4000 dropwise to the cells, gradually increasing the speed. At min 2, allow the centrifuge tube to stand and hold it firmly with both hands. At min 3 and min 4, add 1 mL of RPMI-1640 medium dropwise every 1 min. At min 5 and min 6, add 1 mL of RPMI-1640 medium dropwise every 30 s. At min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s. Then incubate at 37°C for 5 min. Centrifuge at 800 r / min for 10 min, discard the supernatant, gently break up the cells in the centrifuge tube, and add RPMI-1640 selective medium (HAT medium) containing 20% ​​fetal bovine serum and 2% 50×HAT to the medium. Add 200 μL / well to a 96-well cell plate and incubate at 37°C in a 5% CO2 incubator.

[0056] (2) Cell screening and cell line establishment: On day 3 after cell fusion, the fused cells were partially replaced with HAT medium; on day 5, the medium was completely replaced with RPMI-1640 transition medium (HT medium) containing 20% ​​fetal bovine serum and 1% 100×HT; on day 7, the cell supernatant was collected for screening. The screening was carried out in two steps: first, positive cell wells were screened using ic-ELISA; second, the inhibitory effect of teldrone standard on positive cells was determined by ic-ELISA. Cell wells that showed good inhibition of teldrone standard were selected and subcloned using limiting dilution. The same method was used for detection seven days later. Subcloning was performed four times according to the above method to finally obtain the teldrone monoclonal antibody cell line.

[0057] Example 5: Preparation and Identification of Monoclonal Antibodies

[0058] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 2 × 10⁻⁶ g of paraffin oil. 6 Tediroxin hybridoma cells were used. Ascites fluid was collected starting on day 7, and the ascites fluid was purified for antibody purification using the caprylic acid-saturated ammonium sulfate method. Under slightly acidic conditions, caprylic acid precipitates other proteins in the ascites fluid besides IgG immunoglobulins. The precipitate was then discarded by centrifugation. IgG-type monoclonal antibodies were then precipitated with an equal volume of saturated ammonium sulfate solution, centrifuged, and the supernatant was discarded. The precipitate was dissolved in 0.01 M PBS solution (pH 7.4), dialyzed to desalt, and finally the purified monoclonal antibodies were stored at -20°C.

[0059] (1) Coating: The original TDS-EDC-OVA was diluted 3 times with 0.05M (pH 9.6) carbonate buffer starting from 1 µg / mL, 100 μL / well, and reacted at 37℃ for 2 h.

[0060] (2) Washing: Pour off the solution in the plate and wash with washing solution 3 times, 3 minutes each time.

[0061] (3) Sealing: After patting dry, add 200 μL / well sealing solution and react at 37℃ for 2 h. Wash and dry for later use.

[0062] (4) Sample addition: The antiserum (antiserum obtained by diluting the blood from the tail of mice with antibody diluent) was serially diluted from 1:1000 and added to each well of the coating at 100 μL / well. The reaction was carried out at 37℃ for 30 min. After thorough washing, HRP-goat anti-mouse IgG diluted at 1:3000 was added at 100 μL / well. The reaction was carried out at 37℃ for 30 min.

[0063] (5) Color development: Take out the microplate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37°C in the dark for 15 min.

[0064] (6) Termination and measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 value.

[0065] IC50 of the monoclonal antibody tetrodoxin was determined by ic-ELISA. 50 The value was 20 ng / mL, indicating good sensitivity to teldirubin, and it can be used for teldirubin immunoassay.

[0066] The IC50 of monoclonal antibodies against tetrodocin was determined using an indirect competitive ELISA method. 50 The concentration was 20 ng / mL, and its IC50 against compounds such as erythromycin, streptomycin, azithromycin, and spiramycin was verified. 50 And the cross-reactivity rate, the cross-reactivity value is calculated as follows: (Tydrolox IC) 50 IC of other compounds 50 () × 100%, as shown in Table 1.

[0067] Table 1. IC50 of monoclonal antibodies against temaropyrine and its cross-links 50 and cross-reactivity

[0068]

[0069] The monoclonal antibody showed a 100% cross-link against temaroxate, while the cross-links against erythromycin, streptomycin, azithromycin, and spiramycin were all less than 1%. This indicates that the monoclonal antibody obtained in this invention has high sensitivity (IC50) to temaroxate. 50 It has a value of 20 ng / mL and also exhibits high specificity (cross-reactivity with analogs is less than 1%).

[0070] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A hybridoma cell line, characterized in that, The hybridoma cell line was named a monoclonal cell line and its accession number was CGMCC No. 46522.

2. The hybridoma cell line according to claim 1, characterized in that, The hybridoma cell line was obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structural formula of the hapten is shown in Formula I: 。 3. The hybridoma cell line according to claim 2, characterized in that, The complete antigen is obtained by conjugating the hapten to a carrier protein.

4. The hybridoma cell line according to claim 3, characterized in that, The carrier protein includes bovine serum albumin.

5. The use of the hybridoma cell line according to any one of claims 1-4 in the detection of teldiclofenac.

6. A monoclonal antibody secreted by a hybridoma cell line according to any one of claims 1-4.

7. The use of the monoclonal antibody according to claim 6 in the detection of tepidiloxine.

8. A novel testing product for terephthalic acid, characterized in that, The detection product includes the monoclonal antibody as described in claim 6.

9. The testing product according to claim 8, characterized in that, The tested products also include coating agents.

10. The testing product according to claim 9, characterized in that, The coating is prepared from a hapten-coupled carrier protein, wherein the carrier protein includes chicken ovalbumin.

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