A hybridoma cell line secreting tobuterol monoclonal antibody and its application

Abstract

This invention relates to a hybridoma cell line secreting a monoclonal antibody against tobrekterol and its application, belonging to the field of immunoassay technology. The invention involves synthesizing a tobrekterol hapten, preparing a complete tobrekterol antigen, and then screening for a hybridoma cell line secreting a monoclonal antibody against tobrekterol through mouse immunization and cell fusion steps. This hybridoma cell line is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46232. The monoclonal antibody secreted by this hybridoma cell line exhibits good sensitivity and specificity, showing a high IC50 concentration for tobrekterol. 50 The value is 2.72 ng / mL, and the cross-reactivity with various tobbuterol functional analogs is less than 1%. It can be used to prepare tobbuterol detection products. The detection limit for tobbuterol is 0.096 ng / mL, providing an efficient detection method and means for the detection of tobbuterol residues in food.

Description

Technical Field

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

[0002] Beta-adrenergic agonists are used clinically to treat conditions such as bronchial asthma and obstructive pneumonia, and also to treat delayed gestation and abortion in horses and cattle. In the early 1980s, a series of experimental studies found that when beta-adrenergic agonists were used at doses 5-10 times the therapeutic dose, they could enhance fat catabolism and increase protein synthesis in animals such as pigs, cattle, and sheep. Long-term use of feed containing beta-adrenergic agonists could improve feed conversion ratio, increase animal weight and muscle mass, thereby increasing lean meat percentage and playing a role in nutrient redistribution. Therefore, feed or drinking water containing beta-adrenergic agonists is commonly used in livestock production to improve animal growth rate and lean meat percentage; the most common are clenbuterol, ractopamine, and salbutamol. In recent years, with the increase in the types of beta-adrenergic agonists, tobuterol, sematonbuterol, and bambuterol have also been used in animal husbandry to promote growth. Consuming animal-derived foods containing beta-agonists may cause poisoning, manifesting as premature ventricular contractions, tremors of skeletal muscles in the limbs, face, and neck, and metabolic disorders. Although beta-agonists have been banned for many years, the production, sale, and use of clenbuterol-like drugs persist, and strict monitoring and detection of beta-agonists must continue.

[0003] Currently, the relevant standard methods for detecting β-receptor agonists include liquid chromatography-tandem mass spectrometry (LC-MS / MS), gas chromatography-tandem mass spectrometry (GC-MS / MS), high-performance liquid chromatography (HPLC), and colloidal gold immunochromatography. Yue Hanxiao et al. used GC-MS / MS to determine four β-receptor agonists—clenbuterol, brombuterol, tobuterol, and salbutamol—in pork. Pork samples were extracted with ammonium acetate buffer, digested with β-glucuronidase / arylsulfatase, and the digest was purified by centrifugation using an HLB-MCX column in series. Derivatization was then performed using a derivatizing agent, and detection was performed in multiple reaction monitoring (MRM) mode with internal standard quantification. The concentrations of the four β-receptor agonists ranged from 1.76 to 122.00 μg / L. The linear correlation coefficients were all greater than 0.9997 within the concentration range. The limits of detection and quantitation were between 0.13-0.40 μg / kg and 0.40-1.27 μg / kg, respectively. The recoveries at low, medium, and high concentrations were 92.2%-112.8%, with intra-batch coefficients of variation of 1.0%-12.6% and inter-batch coefficients of variation of 1.9%-13.2%. This method utilizes an internal standard to reduce matrix interference and can accurately determine the residues of four β-receptor agonists in pork. However, 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. There is an urgent need to establish a detection method that is simple to operate, highly sensitive, and highly specific. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a tobroodorrol monoclonal antibody and a hybridoma cell line capable of secreting this monoclonal antibody. By synthesizing a tobroodorrol hapten and preparing a complete tobroodorrol antigen, a hybridoma cell line secreting a tobroodorrol monoclonal antibody was obtained through mouse immunization and cell fusion steps. This cell line is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46232.

[0005] The first objective of this invention is to provide a hybridoma cell line that secretes a tobuterol monoclonal antibody, the hybridoma cell line having the accession number CGMCC NO.46232.

[0006] Furthermore, the method for preparing the hybridoma cell line includes the following steps:

[0007] Step S1: Prepare tabuterol hapten, prepare tabuterol immunogen using the obtained tabuterol hapten, combine the obtained tabuterol immunogen with complete Freund's adjuvant to obtain antigen-containing complete Freund's adjuvant, emulsify the obtained tabuterol complete antigen and incomplete Freund's adjuvant to obtain antigen-containing incomplete Freund's adjuvant.

[0008] Step S2: The immunized animals are first immunized with the complete Freund's adjuvant containing the antigen obtained in step S1.

[0009] Step S3: The immunized animals in Step S2 are given a booster immunization with the incomplete Freund's adjuvant containing antigen obtained in Step S1, and then given a sprint immunization with tobutterol immunogen.

[0010] Step S4: Take spleen cells and myeloma cells from the immunized animals that underwent sprint immunization in step S3 and fuse them to obtain the hybridoma cell line.

[0011] Furthermore, the structural formula of the tobrekrol hapten is as follows:

[0012] A second objective of this invention is to provide the application of the above-mentioned hybridoma cell line in the preparation of tobuteromonoclonal antibodies.

[0013] A third objective of this invention is to provide a tobuterol monoclonal antibody, which is obtained from the above-mentioned hybridoma cell line.

[0014] A fourth object of the present invention is to provide a composition comprising the above-described hybridoma cell line and / or the above-described monoclonal antibody.

[0015] A fifth object of the present invention is to provide the use of the above-mentioned hybridoma cell line, the above-mentioned tobrekterol monoclonal antibody, or the above-mentioned composition in the preparation of tobrekterol detection products.

[0016] A sixth object of the present invention is to provide a test strip containing the above-mentioned hybridoma cell line, the above-mentioned tobrekterol monoclonal antibody, or the above-mentioned composition.

[0017] Furthermore, the test strip also includes a sample pad, a conjugate pad, an absorbent pad, and a cellulose acetate membrane. The conjugate pad is coated with tobrekrol monoclonal antibody, which is labeled with colloidal gold or fluorescent microspheres.

[0018] A seventh object of the present invention is to provide a kit containing the above-described hybridoma cell line, the above-described tobuterol monoclonal antibody, or the above-described composition.

[0019] Furthermore, the kit also includes an ELISA plate, tobrekrol-coated antigen, tobrekrol standard solution, enzyme-labeled secondary antibody, and chromogenic solution.

[0020] An eighth object of the present invention is to provide the use of the above-described hybridoma cell line, the above-described monoclonal antibody, the above-described composition, the above-described test strip, or the above-described kit in the detection of tobacterol.

[0021] The beneficial effects of this invention are:

[0022] This invention discloses a hybridoma cell line that secretes monoclonal antibodies against tobrekterol by synthesizing an artificial antigen for tobrekterol. The monoclonal antibody secreted by this cell line exhibits good specificity and detection sensitivity (IC50) for tobrekterol. 50 With a detection limit of 2.72 ng / mL, and cross-reactivity with various tobbuterol functional analogs of less than 1%, it can be used to detect tobbuterol and can also be applied to the preparation of tobbuterol detection products including test strips, kits and biochips. The detection limit for tobbuterol is 0.096 ng / mL, providing raw materials for the immunoassay of tobbuterol residues in livestock and poultry products, and has practical application value.

[0023] Preservation of biological materials

[0024] A monoclonal cell line secreting tobutterol monoclonal antibody, which was deposited on October 31, 2024 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46232, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0025] 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, wherein...

[0026] Figure 1 The standard inhibition curve of the tobuterol monoclonal antibody prepared in the embodiments of the present invention is shown. Detailed Implementation

[0027] 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.

[0028] The culture media involved in the following examples are shown below:

[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] The solutions involved in the following examples are shown below:

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

[0032] Phosphate-buffered saline (PBS): 8.0g NaCl, 0.2g KCl, 0.2g KH2PO4, 2.9g Na2HPO4·12H2O, dissolved in 800mL pure water, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000mL.

[0033] PBST: PBS containing 0.05% Tween 20;

[0034] TMB colorimetric solution: Solution A: 18.43g Na2HPO4·12H2O, 9.33g citric acid, diluted to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution. Mix fresh before use.

[0035] The detection methods involved in the following embodiments are as follows:

[0036] Tobacterol inhibition rate assay: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. The antigen was diluted to 1 μg / mL, 0.3 μg / mL, 0.1 μg / mL, and 0.03 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.3 μg / mL, 0.1 μg / mL, 0.03 μg / mL, and 0.01 μg / mL with antibody dilution buffer. After selecting the optimal operating point, tobacterol standards were diluted to concentrations of 0.11 ng / mL, 0.33 ng / mL, 1 ng / mL, 3 ng / mL, 9 ng / mL, 27 ng / mL, and 81 ng / mL. Following the ic-ELISA procedure, the standard inhibition curves for tobacterol were plotted using Origin 2024, and the IC50 was calculated. 50 and the limit of detection (LOD).

[0037] Example 1: Synthesis of tobrekrol hapten

[0038] The product containing a carboxyl group, namely the hapten TB-hapten, is obtained by reacting to tobutterol technical grade with succinic anhydride.

[0039] The synthesis route is as follows:

[0040]

[0041] The specific steps are as follows:

[0042] Reaction solution A: Tobacterol (50 mg, 0.22 mmol), DMAP (54 mg, 0.44 mmol), and succinic anhydride (44 mg, 0.44 mmol) were weighed and dissolved in pyridine. The solution was refluxed at 100 °C for 48 h with stirring and monitored by TLC. After the reaction was complete, heating was stopped, and the reaction solution was allowed to cool to room temperature. The solution was then concentrated under reduced pressure. 2.0 mL of pure water was added, and the pH was adjusted to approximately 4 with 1.0 mol / L hydrochloric acid. The solution was extracted three times with ethyl acetate and concentrated under reduced pressure. The solution was purified by silica gel column chromatography and concentrated to obtain the hapten TB-hapten (38 mg). The hapten was stored at 4 °C for later use. Yield: 75.0%.

[0043] Example 2: Preparation of tobuteroide complete antigen

[0044] The hapten TB-hapten was conjugated to a carrier protein using the carbodiimide method to obtain the tobbuterol artificial antigen. Specifically, the hapten TB-hapten prepared in Example 1 was conjugated with bovine serum albumin (BSA) to obtain the complete antigen TB-EDC-BSA.

[0045] The preparation method of the complete antigen TB-EDC-BSA is as follows:

[0046] (1) Weigh 2.0 mg of the hapten TB-hapten, 4.3 mg of N-hydroxysuccinimide, and 2.5 mg of 1-ethylcarbodiimide hydrochloride prepared in Example 1, and dissolve them in 300 μL of N,N-dimethylformamide (referred to as solution A). Stir the mixture at room temperature for 4-6 h. Weigh 6.0 mg of BSA (the molar ratio of TB-hapten to BSA is 20:1), add 2 mL of carbonate buffer solution (the dissolved BSA protein is 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 1M NaOH solution, and react at room temperature overnight to obtain the conjugate TB-EDC-BSA.

[0047] (2) Dialysis: Cut an 8cm dialysis bag, boil it in boiling water for 3 minutes and cool it down, and store it in deionized water at 4℃ for later use; put the TB-EDC-BSA conjugate into the dialysis bag and dialyze it in 0.01mol / L PBS, change it every 8 hours, and dialyze for 3 days to obtain the complete antigen TB-EDC-BSA, take it out and store it at -20℃.

[0048] Example 3: Preparation of coating antigen

[0049] Weigh 2.0 mg of the hapten TB-hapten, 4.3 mg of N-hydroxysuccinimide, and 2.5 mg of 1-ethylcarbodiimide hydrochloride prepared in Example 1, and dissolve them in 300 μL of N,N-dimethylformamide (referred to as solution A). Stir at room temperature for 4-6 hours. Weigh 6.0 mg of chicken egg white albumin OVA (TB to OVA molar ratio of 20:1), add 2 mL of carbonate buffer solution (the dissolved OVA protein is referred to as solution B). Under room temperature conditions, add solution A dropwise to solution B, adjust the pH of the mixture to 8-9 with 1M NaOH solution, and react at room temperature overnight to obtain the conjugate TB-EDC-OVA.

[0050] Example 4: Mouse Immunization

[0051] For the initial immunization, BALB / c mice were immunized with a mixture of 100 μg of tobrekrol complete antigen and an equal volume of complete Freund's adjuvant, emulsified, and administered via multiple subcutaneous injections at the nape of the neck. 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 administered 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 administered via intraperitoneal injection. After the third immunization, tail-disconnected blood samples were collected for testing. Serum titers and IC50 were determined using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). 50 Choose high-performance ICs 50 Low-grade mice were fused.

[0052] Example 5: Cell Fusion and Screening

[0053] (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:

[0054] i. 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 × 10⁶ 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.

[0055] ii. After euthanizing 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 the suspension in 50 mL sterile centrifuge tubes, centrifuge at 1200 rpm for 8 minutes, wash the spleen cells with RPMI-1640 medium, remove larger 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.

[0056] iii. 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 rpm 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 at a rate of 200 μL / well to a 96-well cell plate. Incubate at 37°C in a 5% CO2 incubator.

[0057] (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. Screening was performed in two steps: first, positive cell wells were selected using ic-ELISA; second, tobacterol standard was used, and the inhibitory effect on positive cells was determined using ic-ELISA. Cell wells showing good inhibition of tobacterol standard were selected, and subcloning was performed 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 tobacterol monoclonal antibody cell line XCQ.

[0058] Example 6: Preparation and Identification of Monoclonal Antibodies

[0059] 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 Tobacterol hybridoma cells were used, and ascites fluid was collected starting on day 7. The ascites fluid was then purified for antibody 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 0.01M PBS solution (pH 7.4), dialyzed to desalt, and finally the purified monoclonal antibodies were stored at -20°C.

[0060] (1) Coating: The original TB-EDC-OVA was diluted 3 times from 1 μg / mL with 0.05M (pH=9.6) carbonate buffer, 100 μL / well, and reacted at 37℃ for 2 h.

[0061] (2) Washing: Pour out the solution in the plate and wash with washing solution 3 times, 3 minutes each time.

[0062] (3) Sealing: After patting dry, add 200 μL / well sealing solution and react at 37℃ for 2 h. Wash and dry for later use.

[0063] (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 coated sample at 100 μL / well. The mixture was reacted at 37°C for 30 min. After thorough washing, HRP-goat anti-mouse IgG diluted at 1:3000 was added at 100 μL / well. The mixture was reacted at 37°C for 30 min.

[0064] (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.

[0065] (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.

[0066] The IC50 of the monoclonal antibody tobacterol was determined by ic-ELISA. 50 The concentration was 2.72 ng / mL, and the detection limit was 0.096 ng / mL, indicating that it has good sensitivity and specificity for tobacterol and can be used for immunoassay detection of tobacterol.

[0067] Example 7: Application of Monoclonal Antibodies

[0068] The IC50 of the monoclonal antibody against tobbuterol was determined using an indirect competitive ELISA method. 50 The concentration was 2.72 ng / mL, and its IC50 for compounds such as salbutamol, clenbuterol, ractopamine, and mabuterol was verified. 50 and the cross-reactivity rate, the cross-reactivity value is calculated as cross-reactivity rate = (IC50 of tobacterol) 50 IC of other compounds 50 The result is shown in Table 1, calculated as 100% × 100%.

[0069] Table 1. IC50 of monoclonal antibodies against tobacterol and its functional cross-links 50 and cross-reactivity

[0070] IC50 (ng / mL) Cross-reactivity Tobutero 2.72 100％ Salbutamol >1000 <1％ Clentro >1000 <1％ ractopamine >1000 <1％ Mabutro >1000 <1％

[0071] The monoclonal antibody showed 100% cross-reactivity with tobutoperol, while the cross-reactivity rates with salbutamol, clenbuterol, ractopamine, and mabutoperol were all less than 1%. This indicates that the monoclonal antibody obtained in this invention has high sensitivity (IC50) to tobutoperol. 50 It has a value of 2.72 ng / mL and also exhibits high specificity, with a cross-reactivity rate of less than 1% with tobbuterol functional analogues.

[0072] 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 secreting tobuterol monoclonal antibody, characterized in that: The hybridoma cell line has the accession number CGMCC NO.46232.

2. The use of the hybridoma cell line according to claim 1 in the preparation of tobuterol monoclonal antibody.

3. A tobroterol monoclonal antibody, characterized in that: The tobuterol monoclonal antibody is obtained from the hybridoma cell line described in claim 1.

4. The use of the hybridoma cell line of claim 1 or the tobractol monoclonal antibody of claim 3 in the preparation of tobractol detection products.

5. A test strip, characterized in that: The test strip contains the tobuterol monoclonal antibody as described in claim 3.

6. The test strip according to claim 5, characterized in that: The test strip also includes a sample pad, a conjugate pad, an absorbent pad, and a cellulose acetate membrane. The conjugate pad is coated with tobrekrol monoclonal antibody, which is labeled with colloidal gold or fluorescent microspheres.

7. A reagent kit, characterized in that: The kit contains the tobuterol monoclonal antibody as described in claim 3.

8. The reagent kit according to claim 7, characterized in that: The kit also includes an ELISA plate, tobacterol-coated antigen, tobacterol standard solution, enzyme-labeled secondary antibody, and chromogenic solution.

9. The use of the hybridoma cell line of claim 1, the monoclonal antibody of claim 3, or the test strip of claim 5 in the preparation of a tobacterol detection kit.

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