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

Abstract

The invention relates to a hybridoma cell strain secreting a tiletamine 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 monoclonal antibody of the tiletamine is obtained by synthesizing a tiletamine hapten, preparing a tiletamine complete antigen and carrying out mouse immunoreaction and cell fusion steps through screening, and is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC NO.46505. The invention also discloses a preparation method of the hybridoma cell strain capable of secreting the monoclonal antibody of the tiletamine. The monoclonal antibody secreted by the monoclonal antibody has excellent performance and high detection sensitivity to the teletamine, the IC50 value is 0.26 ng / mL, and the cross reaction rate to the teletamine analogue is less than 1%. The antibody and the hybridoma cell strain can be used for preparing a detection product for the teletamine, the detection limit of the obtained detection product is 0.096 ppb, and a novel and convenient analysis tool and method are provided for detection of teletamine residues in biological samples and food.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay, in particular to a hybridoma cell strain secreting tilatamine monoclonal antibody and application thereof. BACKGROUND

[0002] The English common name of tilatamine is Tiletamine (TIL), a dissociative anesthetic, often used in combination with Zolazepam, mainly used for veterinary anesthesia. Tilatamine belongs to phenylcyclohexylpiperidine derivatives, which can inhibit pain signal transmission by blocking NMDA receptors (glutamate receptor subtypes) in the central nervous system, and produce anesthetic, analgesic and hallucinogenic effects, and long-term intake has addictive properties. First, tilatamine may be misused as a drug, especially in the context of recreational drugs, and suspected substances or biological samples (such as urine, blood) need to be screened quickly to crack down on illegal use. Second, tilatamine residues in animal-derived food (such as meat) may endanger human health, and need to be quickly detected to ensure compliance. Therefore, it is necessary to accurately quantify tilatamine in biological and food samples.

[0003] Currently, the main method for detecting tilatamine is instrument detection, and the commonly used methods are gas chromatography, liquid chromatography and gas chromatography-mass spectrometry. Although these chromatography-based methods have high sensitivity and specificity, they have some drawbacks, such as the need for thorough sample purification, high solvent consumption, expensive equipment and skilled technicians. Therefore, a rapid and simple method for detecting tilatamine residues is needed.

[0004] Enzyme-linked immunosorbent assay (ELISA) is a highly efficient, sensitive and rapid detection method. The sample pretreatment is simple, the purification steps are few, the analysis capacity is large, the detection cost is low and the operation is simple, which is suitable for on-site rapid detection of a large number of samples, and therefore has been widely used in drug residue analysis. The premise of using enzyme-linked immunosorbent assay to detect tilatamine is to obtain a monoclonal antibody with high specificity and high sensitivity to tilatamine, therefore, it is crucial to find a method for preparing a monoclonal antibody with high specificity and high sensitivity to tilatamine. The inventors tried to prepare a tilatamine monoclonal antibody by hybridoma cells, but in the process of preparing a hybridoma cell strain secreting a tilatamine monoclonal antibody, how to prepare a tilatamine hapten and a complete antigen, how to make the mouse produce a strong immune effect, and how to make the prepared hybridoma cell strain successfully secrete a tilatamine monoclonal antibody with high specificity and high sensitivity, further research is needed. SUMMARY

[0005] To solve the above technical problems, the application synthesizes tegaserod hapten, prepares tegaserod complete antigen, and through mouse immunization and cell fusion steps, a hybridoma cell strain secreting tegaserod monoclonal antibody is screened and preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC NO. 46505.

[0006] The first object of the application is to provide a hybridoma cell strain secreting tegaserod monoclonal antibody, and the preservation number of the hybridoma cell strain is CGMCC NO. 46505.

[0007] Further, the preparation method of the hybridoma cell strain comprises the following steps:

[0008] Step 1, tegaserod hapten and tegaserod complete antigen are prepared, and the obtained tegaserod complete antigen is emulsified with Freund's adjuvant or incomplete Freund's adjuvant to prepare an immunogen;

[0009] Step 2, the obtained immunogen is injected into BALB / c mice through back subcutaneous injection for multiple immunization, and complete Freund's adjuvant is used for the first immunization and incomplete Freund's adjuvant is used for the booster immunization;

[0010] Step 3, the mouse after the above immunization process is bled, and the serum immune titer and immune inhibition ability of the mouse are detected through indirect ELISA to screen out the mouse with high sensitivity of tegaserod antibody in the serum;

[0011] Step 4, the screened mouse is given a booster immunization through intraperitoneal injection, and the booster immunization uses tegaserod complete antigen without Freund's adjuvant;

[0012] Step 5, the spleen cells and myeloma cells of the BALB / c mouse after the booster immunization are fused, the fused cells are screened and cultured through HAT medium, the positive cell holes are detected through indirect ELISA, and the inhibition effect of the positive cell holes is further determined through indirect competitive ELISA method, the positive cell holes with the best inhibition are subcloned through limited dilution method, and finally the hybridoma cell strain secreting high-sensitivity tegaserod monoclonal antibody is screened out.

[0013] Further, in step 1, the structure of the tegaserod hapten is as follows:

[0014]

[0015] Further, in step 1, the structure of the tegaserod complete antigen is as follows:

[0016]

[0017] In one embodiment of the present application, the first immunization is separated from the booster immunization by one month, the booster immunizations are separated by 21 days, and the booster immunization is separated from the boost immunization by 18-21 days.

[0018] In one embodiment of the present application, the first immunization is separated from the booster immunization by one month, the booster immunizations are separated by 21 days, and the booster immunization is separated from the boost immunization by 18-21 days.

[0019] In one embodiment of the present application, the cell fusion is performed by the polyethylene glycol (PEG4000) method.

[0020] The second object of the present application is to provide the use of the above-mentioned hybridoma cell strain in the preparation of a tiliximab monoclonal antibody.

[0021] The third object of the present application is to provide a tiliximab monoclonal antibody, which is secreted by the above-mentioned hybridoma cell strain.

[0022] The fourth object of the present application is to provide a composition comprising the above-mentioned hybridoma cell strain or the above-mentioned monoclonal antibody.

[0023] The fifth object of the present application is to provide the use of the above-mentioned hybridoma cell strain, the above-mentioned tiliximab monoclonal antibody, or the above-mentioned composition in the preparation of a tiliximab detection product.

[0024] The sixth object of the present application is to provide a test strip comprising the above-mentioned hybridoma cell strain, the above-mentioned tiliximab monoclonal antibody, or the above-mentioned composition.

[0025] Further, the test strip further comprises a sample pad, a conjugate pad, an absorbent pad, and an acetate fiber membrane, the conjugate pad is coated with a tiliximab monoclonal antibody, and the tiliximab monoclonal antibody is labeled with colloidal gold or fluorescent microspheres.

[0026] The seventh object of the present application is to provide a kit comprising the above-mentioned hybridoma cell strain, the above-mentioned tiliximab monoclonal antibody, or the above-mentioned composition.

[0027] Further, the kit further comprises an enzyme-labeled plate, a tiliximab coated antigen, a tiliximab standard solution, an enzyme-labeled secondary antibody, and a color developing solution.

[0028] The eighth object of the present application is to provide the use of the above-mentioned hybridoma cell strain, the above-mentioned tiliximab monoclonal antibody, the above-mentioned composition, the above-mentioned test strip, or the above-mentioned kit in the detection of tiliximab.

[0029] The beneficial effects of the present application are as follows:

[0030] The hybridoma cell strain provided by the application can stably secrete the tilidine monoclonal antibody, the obtained monoclonal antibody has excellent performance, high sensitivity and high specificity for the detection of tilidine, the IC 50 value is 0.26 ng / mL, and the cross-reactivity rate of the tilidine analog is less than 1%. Based on the cell strain and the antibody secreted by the cell strain, a tilidine residue detection product can be further developed, the detection limit of the obtained detection product is 0.096 ppb, which provides a new detection tool and method with high specificity and strong convenience for the trace analysis of tilidine in biological samples and food, and has a wide application prospect.

[0031] Biological material preservation

[0032] The monoclonal cell strain secreting the tilidine monoclonal antibody has been preserved in the China General Microbiological Culture Collection Center on April 17, 2025, the preservation number is CGMCC NO. 46505, and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to make the content of the application more easily understood, the application will be further described in detail below according to specific embodiments of the application and in combination with the drawings, in which:

[0034] Figure 1 The tilidine monoclonal antibody provided by the application is used to draw a standard curve for the inhibition of tilidine. DETAILED DESCRIPTION

[0035] The application will be further described below in combination with the drawings and specific embodiments, so that those skilled in the art can better understand the application and implement the application, but the embodiments are not used as the limitation of the application.

[0036] The culture medium involved in the following embodiments is as follows:

[0037] 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, magnesium sulfate anhydrous 48.84, sodium phosphate monobasic anhydrous 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L-glutamine 300, biotin 0.2, calcium D-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.

[0038] The reagents involved in the following examples are as follows:

[0039] Carbonate buffer solution (CBS): weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, respectively, dissolve in a small amount of double distilled water, mix, add double distilled water to about 800 mL, mix well, adjust the pH value to 9.6, add double distilled water to 1000 mL, store at 4°C for standby use.

[0040] Phosphate buffer solution (PBS): 8.00 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4, 2.9 g of Na2HPO4·12H2O, dissolved in 800 mL of pure water, adjusted to pH 7.2-7.4 with NaOH or HCl, and diluted to 1000 mL;

[0041] PBST: PBS containing 0.05% Tween 20;

[0042] Antibody diluent: PBS containing 0.1% gelatin;

[0043] TMB developing solution: A solution: 18.43 g of Na2HPO4·12H2O, 9.33 g of citric acid, and pure water to 1000 mL; B solution: 60 mg of TMB dissolved in 100 mL of ethylene glycol. A and B solutions are mixed at a ratio of 5:1 to obtain TMB developing solution, which is mixed before use.

[0044] The detection method involved in the following examples is as follows:

[0045] Detection method of inhibition rate of tianeptine: The most suitable antigen and antibody concentration in ic-ELISA was selected by checkerboard test. The antigen was diluted to 0.01, 0.03, 0.1 and 0.3 μg / mL with carbonate buffer solution (CBS), and the antibody was diluted to 0.03, 0.1, 0.3 and 1 μg / mL with antibody diluent. After selecting the optimal working point, tianeptine standard was diluted to 8 concentrations (0, 0.01, 0.05, 0.1, 0.2, 0.5, 5 and 50 ng / mL), and the ic-ELISA operation steps were followed. Finally, the standard inhibition curve of tianeptine was obtained by OriginPro 8.5, and the IC50value was calculated. 50 .

[0046] Example 1: Synthesis of tianeptine hapten

[0047] Since the tianeptine small molecule does not have immunogenicity and cannot stimulate the mouse to produce an immune response, and thus produce antibodies, it is necessary to couple tianeptine to a protein through protein coupling technology to make it immunogenic. The commonly used active groups in protein coupling technology include amino, carboxyl, hydroxyl, and sulfhydryl groups. In view of the fact that the tianeptine molecular structure does not contain these active groups, tianeptine is derivatized.

[0048] Dissolve 50 mg of tianeptine in 1 mL of anhydrous pyridine, add 36.8 mg of carboxymethyl hydroxylamine hydrochloride, and react at 80°C water bath for 6 h in the dark. After the reaction solution is blown dry with nitrogen, 3 mL of methanol is added for redissolution, and 5 mL of pure water is added for mixing. The redissolved solution is extracted with ethyl acetate for 3 times, and the supernatant is combined and blown dry with nitrogen to obtain the tianeptine hapten, the structure of which is shown below:

[0049]

[0050] Example 2: Synthesis of tianeptine complete antigen

[0051] Weigh 8.55 mg of tianeptine hapten and 6.21 mg of N-hydroxysuccinimide (NHS), and dissolve them in 200 μL of N,N-dimethylformamide (DMF) at room temperature for 10 min of stirring reaction; then weigh 10.36 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and add it to the tianeptine hapten solution, and stir at room temperature for 6-8 h for activation. Take 6 mg of keyhole limpet hemocyanin (KLH), add it to 3 mL of 0.01M carbonate buffer solution (CBS), and dissolve it thoroughly. Slowly add the activated hapten to the KLH-dissolved diluent, and stir overnight at room temperature. Then dialyze with 0.01M PBS solution to remove unreacted small molecules, and obtain a relatively pure complete antigen, which is identified by ultraviolet absorption scanning method. The structure of the tianeptine complete antigen is shown below:

[0052]

[0053] Example 3: Synthesis of tegretol hapten

[0054] Dissolve 3.8 mg tegretol hapten and 2.76 mg N-hydroxysuccinimide (NHS) in 200 μL of anhydrous N,N-dimethylformamide (DMF) and stir the reaction at room temperature for 10 min; dissolve 4.6 mg l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in the above solution and stir the reaction at room temperature for 6-8 h to obtain a hapten activation solution; dissolve 6 mg of chicken ovalbumin (OVA) in carbonate buffer solution (CBS); slowly add the hapten activation solution to the protein dilution and stir overnight at room temperature. Then, dialyze the reaction solution against 0.01 M PBS solution to remove unreacted small molecules and obtain the hapten.

[0055] Example 4: Preparation of hybridoma cell lines secreting tegretol monoclonal antibodies

[0056] (1) Obtaining of animal immunization

[0057] Mix and emulsify the tegretol complete antigen with an equal amount of Freund's adjuvant and subcutaneously inject the BALB / c mice at multiple points on the nape (except for the booster immunization); the first immunization uses complete Freund's adjuvant at a dose of 100 μg per mouse; the multiple booster immunizations use incomplete Freund's adjuvant at a dose of 50 μg per mouse; the booster immunization uses physiological saline diluted directly without adjuvant and is injected intraperitoneally at a dose of 25 μg per mouse; the interval between the first immunization and the second booster immunization is one month, the interval between the multiple booster immunizations is 21 days, and the interval between the booster immunization and the last booster immunization is 18-21 days; observe the immunization effect of the mice by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect the titer and inhibition of the mouse serum.

[0058] (2) Cell fusion

[0059] Three days after the booster immunization, perform cell fusion according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, and the specific steps are as follows:

[0060] a. Take blood from the tail, immediately sterilize the mouse in 75% alcohol for about 5 min, and then take out the mouse's spleen under sterile conditions, gently grind the spleen with a syringe rubber head, and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension, collect, centrifuge (1200 rpm, 8 min), wash the spleen cells with RPMI-1640 culture medium three times, and then dilute the spleen cells to a certain volume, count, and reserve;

[0061] b, collection of SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells are expanded in 10% FBS (fetal bovine serum) RPMI-1640 medium in a 5% CO2 incubator, and the number of SP2 / 0 tumor cells is required to reach 1-4 x 10 7 , to ensure that the SP2 / 0 tumor cells are in the logarithmic growth phase before fusion, and to collect the tumor cells and suspend them in RPMI-1640 base medium for cell counting before fusion;

[0062] c, fusion process 7 min: 1 mL of PEG 4000 is added to the cells from slow to fast in the first minute; 2 min, stand; 3 min and 4 min, add 1 mL of RPMI-1640 medium within 1 min; 5 min and 6 min, add 2 mL of RPMI-1640 medium within 1 min; 7 min, add 1 mL of RPMI-1640 medium every 10 seconds. Except for the 2nd minute, the solution is constantly shaken at other times. Then 37°C incubation for 5 min; centrifugation (800 rpm, 8 min), discard the supernatant, resuspend in RPMI-1640 screening medium containing 20% fetal bovine serum, 2% 50x HAT, add 200 μL / well to a 96-well cell plate, and incubate in a 37°C, 5% CO2 incubator.

[0063] (3) Cell fusion and cell line establishment

[0064] On the 3rd day after cell fusion, the fusion cells are subjected to RPMI-1640 screening medium semi-replacement, and on the 5th day, the cells are subjected to full replacement with RPMI-1640 transition medium containing 20% fetal bovine serum, 1% 100x HT, and on the 7th day, the cell supernatant is screened.

[0065] The screening is divided into two steps: first, positive cell wells are screened by ic-ELISA method, and second, teicoplanin is used as a standard for ic-ELISA method to determine the inhibition effect of positive cells.

[0066] The cell wells with good inhibition to teicoplanin standard are selected, and subcloning is performed by limiting dilution method, and after 7 days, the same method is used for detection.

[0067] At least three times of subcloning are performed according to the above method, and finally the teicoplanin monoclonal antibody cell line is obtained.

[0068] Example 5: Preparation and identification of teicoplanin monoclonal antibody

[0069] Take 8-10 week old BALB / c mice, and inject 1 mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, inject 1 x 10 6Starting from day 7, ascites fluid was collected from telexamine hybridoma cells, and the ascites fluid was purified for antibody using the caprylic acid-saturated ammonium sulfate method.

[0070] Under slightly acidic conditions, octanoic acid can precipitate other proteins in the ascites fluid besides IgG immunoglobulin. After centrifugation, the precipitate is discarded. Then, an equal volume of saturated ammonium sulfate solution is used to precipitate IgG-type monoclonal antibodies. After centrifugation, the supernatant is discarded. The antibodies are dissolved in 0.01M PBS solution (pH=7.4), dialyzed to desalt, and finally purified monoclonal antibodies are obtained and stored at -20℃.

[0071] The IC50 of teletamine monoclonal antibody was measured using an indirect competitive ELISA. 50 The value is 0.26 ng / mL (e.g. Figure 1 As shown in the figure, this indicates that it has good sensitivity to teletamine and can be used for teletamine immunoassay detection.

[0072] Verify its IC for similar products 50 and cross-reactivity rate, with a cross-reactivity rate of less than 1% for related analogues, where cross-reactivity rate = (IC value of telastatin) 50 ICs of similar types 50 The cross-reactivity rate (CRR) is calculated as 100%, indicating that this antibody has high sensitivity and specificity to telexamine, as shown in Table 1.

[0073] Table 1. IC50 of monoclonal antibodies against teretamine and analogues 50 and cross-reactivity rate

[0074] IC 50 (ng / mL) Cross-reactivity (%) Tilidine 0.26 100 Fluoro- ketone 50.12 ＜1 Metomidate 345.21 ＜1 Propofol 231.25 ＜1 Etomidate 125.22 ＜1 Olanzapine 552.12 ＜1

[0075] Example 6: Application of teretamine monoclonal antibody

[0076] The monoclonal antibody prepared from hybridoma cell lines via in vivo ascites fluid was used in a thiamethoxam ELISA addition and recovery assay. The specific steps are as follows:

[0077] (1) Coat a 96-well microplate with 0.1 μg / mL of the coating stock diluted with carbonate buffer (CBS), 100 μL per well, dry at 37°C for 2 h, wash the plate three times with PBST washing buffer, 200 μL per well each time, for 3 min each time, and pat dry.

[0078] (2) Block with CBS containing 0.2% gelatin, 200 μL per well, dry at 37°C for 2 h, wash the plate three times with PBST washing solution, 200 μL per well each time, 3 min each time, and pat dry;

[0079] (3) 0, 0.01, 0.05, 0.1, 0.2, 0.5, 5 and 50 ng / mL of the tiacumumab standard solution was prepared with phosphate buffered saline (PBS) respectively, and the standard solution and the sample extract to be detected were added to the blocked enzyme-labeled plate, 50 μL per well, 3 repeated holes for each sample, then 50 μL of tiacumumab monoclonal antibody diluted to 0.1 μg / mL was added per well, 37°C reaction for 30 min, then the plate was washed and dried;

[0080] (4) 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 with PBS containing 0.1% gelatin was added per well, 37°C reaction for 30 min, then the plate was washed and dried;

[0081] (5) 100 μL of TMB color developing liquid was added per well, 37°C color development for 15 min, then 50 μL of 2M H2SO4 stop solution was added per well, and the absorbance value was measured at 450 nm;

[0082] (6) Recovery.

[0083] Urine was selected as the detection sample.

[0084] 0.01 ppb, 0.1 ppb and 1 ppb of tiacumumab standard was added to the sample (according to the linear range of the antibody and the IC 50 The set concentration was added, and then mixed on an electric shaker. The indirect competitive ELISA was used for the recovery test, and the recovery rates were 89%, 84% and 92% respectively. The detection range was 0.048-1.44 ppb, and the limit of detection (LOD) was 0.096 ppb.

[0085] Obviously, the above examples are only examples for the sake of clarity, and are not limited to the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A hybridoma cell line secreting tiletamine monoclonal antibody, characterized in that: The hybridoma cell line has the accession number CGMCC NO.46505.

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

3. A teletamine monoclonal antibody, characterized in that: The teletamin monoclonal antibody is secreted by the hybridoma cell line described in claim 1.

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

5. The use of the hybridoma cell line of claim 1, the teletamine monoclonal antibody of claim 3, or the composition of claim 4 in the preparation of teletamine detection products.

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

7. The test strip according to claim 6, 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 teletamine monoclonal antibody, which is labeled with colloidal gold or fluorescent microspheres.

8. A reagent kit, characterized in that: The kit contains the hybridoma cell line of claim 1, the teletamine monoclonal antibody of claim 3, or the composition of claim 4.

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

10. The use of the hybridoma cell line of claim 1, the teletamine monoclonal antibody of claim 3, the composition of claim 4, the test strip of claim 6, or the kit of claim 8 in the detection of teletamine.

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