Hybridoma cell strain secreting tubuterol monoclonal antibody and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain capable of secreting a tubuterol monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of immunodetection. According to the invention, a hybridoma cell strain capable of secreting the tolbuterol monoclonal antibody is obtained by synthesizing a tolbuterol hapten, preparing a tolbuterol complete antigen, and carrying out mouse immunoreaction and cell fusion, and is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC NO.46232. The invention further discloses a preparation method of the tolbuterol monoclonal antibody. The monoclonal antibody secreted by the hybridoma cell strain has good sensitivity and specificity, the IC50 value of the monoclonal antibody to tubuterol is 2.72 ng / mL, the cross reaction rate of the monoclonal antibody to various tubuterol functional analogues is smaller than 1%, the monoclonal antibody can be applied to preparation of tubuterol detection products, the detection limit of the monoclonal antibody to tubuterol is 0.096 ng / mL, and the monoclonal antibody can be applied to detection of tubuterol. And an efficient detection method and means are provided for the detection of tobraterol residues in food.

Description

Technical Field

[0001] The present invention relates to the technical field of immune detection, in particular to a hybridoma cell strain secreting Tubuterol monoclonal antibody and application thereof. Background Art

[0002] β-receptor agonists can be used in clinical medicine to treat bronchial asthma, obstructive pneumonia and other diseases. They can also be used to treat late birth and miscarriage in horses and cattle. In the early 1980s, a series of experimental studies found that when the dose of β-receptor agonists was 5-10 times the therapeutic dose, the fat decomposition metabolism of pigs, cattle, sheep and other animals could be enhanced, and the protein synthesis rate could be increased. If feed containing β-receptor agonists was used for a long time, the feed conversion rate could be improved, and the weight and muscle tissue content of animals could be increased to increase the lean meat rate and play a role in nutrient redistribution. Therefore, feed or drinking water containing β-receptor agonists is often used in animal husbandry production to increase the growth rate and lean meat rate of animals; the most common ones are clenbuterol, ractopamine and salbutamol. In recent years, with the increase in the types of β-receptor agonists, tolbuterol, semanterol, bambuterol, etc. have also been used in animal husbandry to promote growth. When people consume animal-derived foods containing beta-agonists, they may suffer from poisoning reactions, including premature ventricular beats, tremors in the limbs, face, and neck, and metabolic disorders. Although beta-agonists have been banned for many years, the testing and monitoring of beta-agonists still needs to be strictly enforced.

[0003] At present, the relevant standard methods used for the detection of β-receptor agonists include liquid chromatography-tandem mass spectrometry, gas chromatography-tandem mass spectrometry, high performance liquid chromatography, colloidal gold immunochromatography and other detection methods. Yue Hanxiao et al. used gas chromatography-tandem mass spectrometry to determine four β-receptor agonists, clenbuterol, bromobuterol, tolbuterol and salbutamol, in pork. The pork samples were extracted with ammonium acetate buffer, hydrolyzed with β-glucuronidase / arylsulfatase, and the hydrolyzate was centrifuged and purified by HLB-MCX columns in series. Then, it was derivatized with a derivatizing agent and detected in the multiple reaction monitoring mode (MRM) mode. The internal standard method was used for quantification. The four β-receptor agonists were in the range of 1.76-122.00 μg / L. The linear correlation coefficients within the concentration range were all greater than 0.9997, the detection limits and quantification limits were between 0.13-0.40 μg / kg and 0.40-1.27 μg / kg, respectively, the recovery rates at low, medium and high concentrations were 92.2%-112.8%, the intra-batch coefficient of variation was 1.0%-12.6%, and the inter-batch coefficient of variation was 1.9%-13.2%; this method can reduce matrix interference by using the internal standard method and can accurately determine the residues of four β-receptor agonists in pork. These methods are complicated to operate and the sample processing is cumbersome, which is not conducive to the detection of large batches of samples in actual production. It is urgent to establish a detection method that is simple to operate, highly sensitive and highly specific. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a monoclonal antibody of Tubuterol and a hybridoma cell line capable of secreting the monoclonal antibody. Tubuterol complete antigen is prepared by synthesizing Tubuterol hapten, and after mouse immune response and cell fusion steps, a hybridoma cell line secreting Tubuterol monoclonal antibody is screened and obtained, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Committee with a deposit number of CGMCC NO.46232.

[0005] The first object of the present invention is to provide a hybridoma cell strain secreting tolbutrol monoclonal antibody, wherein the deposit number of the hybridoma cell strain is CGMCC NO.46232.

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

[0007] Step S1, preparing Tubuterol hapten, using the obtained Tubuterol hapten to prepare Tubuterol immunogen, mixing the obtained Tubuterol immunogen with complete Freund's adjuvant to obtain antigen-containing complete Freund's adjuvant, and emulsifying the obtained Tubuterol complete antigen and incomplete Freund's adjuvant to obtain antigen-containing incomplete Freund's adjuvant;

[0008] Step S2, using the antigen-containing complete Freund's adjuvant obtained in step S1 to perform the first immunization on the immunized animal;

[0009] Step S3, the immunized animals immunized in step S2 are boosted with the incomplete Freund's adjuvant containing the antigen obtained in step S1, and sprint immunized with the Tubuterol immunogen;

[0010] Step S4, taking the spleen cells of the immunized animal subjected to sprint immunization in step S3 and performing cell fusion with myeloma cells to obtain the hybridoma cell line;

[0011] Furthermore, the structural formula of the Tubuterol hapten is:

[0012] The second object of the present invention is to provide the use of the hybridoma cell line in the preparation of Tubuterol monoclonal antibody.

[0013] The third object of the present invention is to provide a Tubuterol monoclonal antibody, which is secreted by the above hybridoma cell line.

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

[0015] The fifth object of the present invention is to provide the use of the hybridoma cell line, the Tubuterol monoclonal antibody or the composition in the preparation of a Tubuterol detection product.

[0016] The sixth object of the present invention is to provide a test strip, wherein the test strip contains the hybridoma cell line, the tolbutrol monoclonal antibody or the combination

[0017] Furthermore, the test strip also includes a sample pad, a conjugation pad, a water absorbent pad and a cellulose acetate membrane, the conjugation pad is coated with a Tubuterol monoclonal antibody, and the Tubuterol monoclonal antibody is labeled with colloidal gold or fluorescent microspheres.

[0018] The seventh object of the present invention is to provide a kit, which contains the above hybridoma cell line, the above Tubuterol monoclonal antibody or the above composition.

[0019] Furthermore, the kit also includes an ELISA plate, a Tubutrol coated antigen, a Tubutrol standard solution, an enzyme-labeled secondary antibody and a color developing solution.

[0020] The eighth object of the present invention is to provide the use of the hybridoma cell line, the monoclonal antibody, the composition, the test strip or the kit in the detection of Tubuterol.

[0021] Beneficial effects of the present invention:

[0022] The present invention prepares a hybridoma cell line secreting a monoclonal antibody against Tubuterol by synthesizing an artificial Tubuterol antigen. The monoclonal antibody secreted by the hybridoma cell line has good specificity and detection sensitivity (IC 50 The value is 2.72ng / mL), and the cross-reaction rate to various Tubuterol functional analogues is less than 1%, which can realize the detection of Tubuterol. It can also be used to prepare Tubuterol detection products including test strips, kits and biochips. The detection limit of Tubuterol is 0.096ng / mL, which provides raw materials for the immunological detection of Tubuterol residues in livestock and poultry products and has practical application value.

[0023] Biomaterial Deposit

[0024] A monoclonal cell line secreting tolbutrol monoclonal antibody, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection on October 31, 2024, with the deposit number CGMCC NO.46232, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0026] Figure 1 This is the standard inhibition curve of the Tubuterol monoclonal antibody prepared in the example of the present invention. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0028] The culture medium involved in the following examples is as follows:

[0029] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine 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, p-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, niacinamide 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 as follows:

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

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

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

[0034] TMB colorimetric solution: Solution A: Na2HPO 4. 12H2O 18.43g, citric acid 9.33g, pure water to 1000mL; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solution A and solution B in a ratio of 5:1 to obtain TMB colorimetric solution, which should be mixed before use.

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

[0036] Tubuterol inhibition rate detection method: The most suitable antigen and antibody concentrations in ic-ELISA are selected through the checkerboard test. The antigen is diluted to 1μg / mL, 0.3μg / mL, 0.1μg / mL, 0.03μg / mL with carbonate buffer (CBS), and the antibody is diluted to 0.3μg / mL, 0.1μg / mL, 0.03μg / mL, 0.01μg / mL with antibody diluent. After selecting the optimal working point, dilute the Tubuterol standard to concentrations of 0.11ng / mL, 0.33ng / mL, 1ng / mL, 3ng / mL, 9ng / mL, 27ng / mL, 81ng / mL, etc., follow the ic-ELISA operating steps, and finally use Origin2024 to draw a graph to obtain the Tubuterol standard inhibition curve and calculate the IC 50 and limit of detection (LOD).

[0037] Example 1: Synthesis of Tubuterol Hapten

[0038] The product having a carboxyl group, namely the hapten TB-hapten, is obtained by reacting the original drug of Tubuterol with succinic anhydride.

[0039] The synthetic route is as follows:

[0040]

[0041] The specific steps are:

[0042] Reaction A solution: weigh Tubuterol (50 mg, 0.22 mmol), DMAP (54 mg, 0.44 mmol) and succinic anhydride (44 mg, 0.44 mmol) and dissolve in pyridine, stir and reflux at 100°C for 48 h, and monitor by TLC. When the reaction is complete, stop heating, wait for the temperature of the reaction solution to drop to room temperature, and concentrate under reduced pressure; add 2.0 mL of pure water, adjust the pH to about 4 with 1.0 mol / L hydrochloric acid, extract with ethyl acetate three times, and concentrate under reduced pressure; purify by silica gel column chromatography, concentrate, and obtain hapten TB-hapten (38 mg), which is stored at 4°C for future use, with a yield of 75.0%.

[0043] Example 2: Preparation of Tubuterol Complete Antigen

[0044] The hapten TB-hapten was coupled with the carrier protein by the carbodiimide method to obtain the Tubuterol artificial antigen. The hapten TB-hapten prepared in Example 1 was coupled 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 prepared in Example 1, 4.3 mg of N-hydroxysuccinimide, and 2.5 mg of 1-ethylcarbodiimide hydrochloride, dissolve them in 300 μL of N,N-dimethylformamide (referred to as solution A), and react at room temperature for 4-6 hours with stirring. Weigh 6.0 mg of BSA (the molar ratio of TB-hapten to BSA is 20:1), add 2 mL of carbonate buffer solution (referred to as solution B after the BSA protein is dissolved), 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 at room temperature overnight to obtain the conjugate TB-EDC-BSA.

[0047] (2) Dialysis: 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°C for later use; place the conjugate TB-EDC-BSA conjugate solution in the dialysis bag and dialyze it in 0.01 mol / L PBS, changing it every 8 h. After 3 days of dialysis, the complete antigen TB-EDC-BSA is obtained, which is then removed and stored at -20°C.

[0048] Example 3: Preparation of coated antigen

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

[0050] Example 4: Immunization of mice

[0051] For the first immunization, the complete antigen of Tubuterol was mixed with an equal amount of complete Freund's adjuvant and emulsified at a dose of 100 μg / mouse for injection, and BALB / c mice were immunized by multiple subcutaneous injections on the back of the neck; after a lapse of 4 weeks, a booster immunization was performed, and the dose of the complete antigen was halved (50 μg / mouse) and mixed with incomplete Freund's adjuvant for emulsification, and the intervals between subsequent booster immunizations were 3 weeks; for the sprint immunization, the dose was halved again (25 μg / mouse), and the complete antigen was diluted with saline and injected intraperitoneally into the mice. After the third immunization, the mice were tail-cut and blood was collected for testing, and the titer and IC of the mouse serum were tested by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). 50 , select high titer, IC 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. Collect 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. Before fusion, the number of SP2 / 0 tumor cells should reach 1-4×10 7 , to ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. When fusion occurs, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counts are performed;

[0055] ii. After killing the mouse by dislocating the neck, immediately sterilize it in 75% alcohol and soak it for about 5 minutes. Remove the spleen of the mouse by aseptic operation, grind it moderately with a syringe rubber tip and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect it in a 50mL sterile centrifuge tube and centrifuge it at 1200r / min for 8 minutes. Wash the spleen cells with RPMI-1640 medium, pick out larger tissue impurities, and repeat the operation three times. After the last centrifugation, dilute the spleen cells to a certain volume, count them, and set aside;

[0056] iii. Fusion process 7min: In the first minute, 1mL of PEG 4000 was added to the cells from slow to fast speed; in the second minute, the tube was left to stand and held tightly with both hands. In the third and fourth minutes, 1mL of RPMI-1640 medium was added within 1min; in the fifth and sixth minutes, 1mL of RPMI-1640 medium was added every 30s; in the seventh minute, 1mL of RPMI-1640 medium was added every 10s. Then, the cells were incubated at 37°C for 5min. Centrifuged at 800r / min for 10min, the supernatant was discarded, the cells in the centrifuge tube were gently knocked apart, and RPMI-1640 selective medium (HAT medium) containing 20% ​​fetal bovine serum and 2% 50×HAT was added thereto, and added to a 96-well cell plate at 200μL / well, and cultured in a 37°C, 5% CO2 incubator.

[0057] (2) Cell screening and cell line establishment: On the third day after cell fusion, half of the fused cells were replaced with HAT medium; on the fifth day, the medium was completely replaced with RPMI-1640 transition medium (HT medium) containing 20% ​​fetal bovine serum and 1% 100×HT; on the seventh day, the cell supernatant was taken for screening. The screening was divided into two steps: the first step was to screen the positive cell wells by ic-ELISA, and the second step was to select the tubutrol standard and measure the inhibitory effect of the positive cells by ic-ELISA. The cell wells with better inhibition of the tubutrol standard were selected, subcloned by limiting dilution method, and tested by the same method seven days later. Four subclonings were performed according to the above method, and finally the tubutrol monoclonal antibody cell line TB was obtained.

[0058] Example 6: Preparation and identification of monoclonal antibodies

[0059] 8-10 week old BALB / c mice were intraperitoneally injected with 1 mL of sterile paraffin oil. Seven days later, 2 × 10 6 Tubuterol hybridoma cells, ascites was collected from the seventh day, and the ascites was purified by the caprylic acid-saturated ammonium sulfate method. Under acidic conditions, caprylic acid can precipitate other miscellaneous proteins except IgG immunoglobulin in the ascites, and then centrifuge and discard the precipitate; then use an equal amount of saturated ammonium sulfate solution to precipitate IgG type monoclonal antibodies, centrifuge, discard the supernatant, dissolve with 0.01M PBS solution (pH 7.4), dialyze and desalt, and finally obtain the purified monoclonal antibody and store it at -20℃.

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

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

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

[0063] (4) Sample addition: dilute the antiserum (blood was collected from the mouse tail and diluted with antibody diluent to the corresponding multiples) starting from 1:1000 and add it to the coated wells of each dilution, 100 μL / well, and react at 37°C for 30 min. After thorough washing, add 1:3000 diluted HRP-goat anti-mouse IgG, 100 μL / well, and react at 37°C for 30 min.

[0064] (5) Color development: Take out the ELISA plate, 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 an ELISA reader. 450 value.

[0066] Determination of IC of monoclonal antibody Tubuterol by IC-ELISA 50 It is 2.72ng / mL, and the detection limit is 0.096ng / mL, indicating that it has good sensitivity and specificity for Tubuterol and can be used for Tubuterol immunoassay detection.

[0067] Example 7: Application of monoclonal antibodies

[0068] The IC of monoclonal antibodies against Tubuterol was determined using an indirect competitive ELISA method. 50 The IC values ​​of salbutamol, clenbuterol, ractopamine, mabuterol and other compounds were verified. 50 and cross-reaction rate. The cross-reaction value was calculated as cross-reaction rate = (IC 50 / IC of other compounds 50 )×100%, the results are shown in Table 1.

[0069] Table 1 IC of monoclonal antibodies against Tubuterol and functional cross-products 50 and cross-reaction rate

[0070] IC50(ng / mL) Cross-reaction rate Tubuterol 2.72 100％ Salbutamol >1000 <1％ Clenbuterol >1000 <1％ Ractopamine >1000 <1％ Mabutero >1000 <1％

[0071] The cross-reaction rate of the monoclonal antibody to Tubuterol is 100%, and the cross-reaction rate to salbutamol, clenbuterol, ractopamine, and mabuterol is less than 1%. This indicates that the monoclonal antibody obtained by the present invention has a high sensitivity to Tubuterol (IC 50 The value was 2.72 ng / mL), and it also had high specificity, with a cross-reaction rate of less than 1% to the functional analogues of Tubuterol.

[0072] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A hybridoma cell line secreting a monoclonal antibody tobutrol, characterized in that: The deposit number of the hybridoma cell line is CGMCC NO.46232.

2. Use of the hybridoma cell line according to claim 1 in the preparation of tolbutrol monoclonal antibodies.

3. A tolbutrol monoclonal antibody, characterized in that: The Tubuterol monoclonal antibody is secreted by the hybridoma cell line according to claim 1.

4. A composition, characterized in that: The composition comprises the hybridoma cell line according to claim 1 and / or the monoclonal antibody according to claim 3.

5. Use of the hybridoma cell line according to claim 1, the Tubuterol monoclonal antibody according to claim 3 or the composition according to claim 4 in preparing a Tubuterol detection product.

6. A test strip, characterized in that: The test strip contains the hybridoma cell line according to claim 1, the tolbutrol monoclonal antibody according to claim 3 or the composition according to claim 4.

7. The test strip according to claim 6, characterized in that: The test strip also includes a sample pad, a conjugation pad, a water-absorbing pad and a cellulose acetate membrane. The conjugation pad is coated with a Tubuterol monoclonal antibody, and the Tubuterol monoclonal antibody is labeled with colloidal gold or fluorescent microspheres.

8. A kit, characterized in that: The kit contains the hybridoma cell line according to claim 1, the tolbutrol monoclonal antibody according to claim 3 or the composition according to claim 4.

9. The kit according to claim 8, characterized in that: The kit also includes an ELISA plate, a Tubutrol coated antigen, a Tubutrol standard solution, an enzyme-labeled secondary antibody and a color developing solution.

10. Use of the hybridoma cell line according to claim 1, the monoclonal antibody according to claim 3, the composition according to claim 4, the test strip according to claim 6 or the kit according to claim 8 in the detection of Tulbuterol.

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