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

Abstract

The invention relates to a hybridoma cell strain capable of secreting a Telemol monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of immunodetection. According to the invention, 3, 5-dinitrosalicylic acid is used as a hapten to prepare a complete antigen for immunizing a host, and a hybridoma cell strain secreting the monoclonal antibody of the telolenol is obtained by screening and is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCCNO.46230. The invention further discloses a preparation method of the monoclonal antibody of the telolenol. The monoclonal antibody secreted by the hybridoma has relatively high specificity and detection sensitivity to the telolenol, the IC50 value is 2.14 ng / mL, and the monoclonal antibody has no cross reaction with a structural analogue of the telolenol, can be applied to preparation of a telolenol detection product, and provides a powerful tool for related research and application.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a hybridoma cell line secreting the monoclonal antibody of tecnazene and its application. Background Art

[0002] Tecnazene belongs to dinitrophenol herbicides and is a contact herbicide that affects the respiration and photosynthesis of mitochondria and chloroplasts. In mitochondria, at low concentrations, it acts as an uncoupler of oxidative phosphorylation; at higher concentrations, it may inhibit the electron transport chain before cytochrome c. It is commonly used to control the emergence of annual broadleaf weeds in various crops. The structure of dinitrophenol compounds is extremely dangerous even at very low concentrations, especially for animals, and can cause developmental toxicity. It is toxic when in contact with the skin and extremely poisonous if swallowed. Due to the long persistence of tecnazene, it can accumulate in the soil. In addition, it may cause long-term adverse effects in the aquatic environment. In China, the maximum residue limit of tecnazene is 0.01 mg / kg. Detecting the presence of tecnazene residues in agricultural products is crucial for ensuring human health.

[0003] Currently, the main methods for detecting tecnazene are instrumental detections, commonly including high performance liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS / MS), high performance liquid chromatography mass spectrometry (HPLC-MS), ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS). For example, Patent CN114487224A discloses a method for detecting acidic pesticides in fruits and vegetables, which uses solid phase extraction-high performance liquid chromatography-tandem mass spectrometry to qualitatively and quantitatively detect and analyze 12 kinds of acidic pesticide residues in fruits and vegetables. Although these chromatography-based methods have high sensitivity and specificity, there are some disadvantages, such as the need for thorough sample purification, high solvent consumption, expensive equipment, and skilled technicians. Therefore, there is a need for a rapid and simple method for detecting tecnazene residues.

[0004] Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method. During detection, the pretreatment of samples is simple, the purification steps are few, the analysis capacity is large, the detection cost is low, and the operation is simple. It is suitable for rapid on-site detection of a large number of samples, so it has been widely used in drug residue analysis. The premise of using the enzyme-linked immunosorbent assay to detect dinoterb is to obtain a monoclonal antibody with high specificity and high sensitivity to dinoterb. Therefore, it is very crucial to find a method for preparing a monoclonal antibody with high specificity and high sensitivity to dinoterb. The inventor tried to prepare a dinoterb monoclonal antibody through hybridoma cells. However, in the process of preparing a hybridoma cell line that can secrete a dinoterb monoclonal antibody, how to prepare a dinoterb hapten and a complete antigen, how to make mice produce a strong immune effect, how to make the prepared hybridoma cell line successfully secrete a dinoterb monoclonal antibody, and how to make the secreted dinoterb monoclonal antibody have strong specificity and high sensitivity still require further research. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a hybridoma cell line that secretes a dinoterb monoclonal antibody and its application. The present invention uses 3,5-dinitrosalicylic acid as a hapten to prepare a complete antigen for immunizing a host, and screens out a hybridoma cell line that secretes a dinoterb monoclonal antibody, which is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC NO.46230.

[0006] A hybridoma cell line that secretes a dinoterb monoclonal antibody, and the preservation number of the hybridoma cell line is CGMCC NO.46230.

[0007] Furthermore, the preparation method of the above hybridoma cell line includes the following steps:

[0008] Step S1: Prepare dinoterb hapten 1 and dinoterb complete antigen, and emulsify the obtained dinoterb complete antigen with Freund's adjuvant or incomplete Freund's adjuvant to prepare an immunogen.

[0009] Step S2: Inject the obtained immunogen subcutaneously into the back of BALB / c mice for multiple immunizations. Complete Freund's adjuvant is used for the first immunization, and incomplete Freund's adjuvant is used for booster immunizations.

[0010] Step S3: Prepare dinoterb hapten 2 and dinoterb coating antigen, collect blood from the mice that have undergone the above immunization process, detect the immune titer and immune inhibition ability of mouse serum by indirect ELISA, and screen out the mice with high sensitivity of dinoterb antibodies in the serum.

[0011] Step S4: Boost immunize the screened mice by intraperitoneal injection, and the booster immunization uses a dinoterb complete antigen without Freund's adjuvant.

[0012] Step S5: Fuse the splenocytes of BALB / c mice after booster immunization with myeloma cells. The fused cells are screened and cultured in HAT medium. Positive cell wells are detected by indirect ELISA, and the inhibitory effect of positive cell wells is further determined by indirect competitive ELISA. Subcloning of the positive cell wells with the best inhibition is performed by the limiting dilution method, and finally a hybridoma cell line capable of secreting high-sensitivity tebutam monoclone antibody is screened out.

[0013] Further, the structural formula of the tebutam hapten 1 is

[0014] Further, the structural formula of the tebutam hapten 2 is

[0015]

[0016] The second object of the present invention is to provide the application of the above hybridoma cell line in the preparation of tebutam monoclonal antibody.

[0017] The third object of the present invention is to provide a tebutam monoclonal antibody, which is secreted by the above hybridoma cell line.

[0018] The fourth object of the present invention is to provide a composition, which comprises the above hybridoma cell line and / or the above monoclonal antibody.

[0019] The fifth object of the present invention is to provide the application of the above hybridoma cell line, the above tebutam monoclonal antibody or the above composition in the preparation of tebutam detection products.

[0020] Further, the tebutam detection products include reagents, test strips, kits or biochips.

[0021] The sixth object of the present invention is to provide a test strip, which contains the above hybridoma cell line, the above tebutam monoclonal antibody or the above composition.

[0022] Further, the test strip further includes a sample pad, a conjugate pad, an absorbent pad and a cellulose acetate membrane. The conjugate pad is coated with tebutam monoclonal antibody, and the tebutam monoclonal antibody is labeled with colloidal gold or fluorescent microspheres.

[0023] The seventh object of the present invention is to provide a kit, which contains the above hybridoma cell line, the above tebutam monoclonal antibody and the above composition.

[0024] Furthermore, the kit further includes an enzyme-linked immunosorbent assay (ELISA) plate, the antigen coated with ternbutryn, the ternbutryn standard solution, the enzyme-labeled secondary antibody, and the chromogenic solution. In one embodiment of the present invention, the enzyme-labeled secondary antibody is labeled with horseradish peroxidase.

[0025] In one embodiment of the present invention, the chromogenic solution is a TMB chromogenic solution.

[0026] The eighth object of the present invention is to provide the application of the above hybridoma cell line, the above ternbutryn monoclonal antibody, the above composition, the above test strip or the above kit in the detection of ternbutryn.

[0027] Advantages of the present invention:

[0028] The hybridoma cell line provided by the present invention can secrete ternbutryn monoclonal antibody, providing a stable antibody source for the specific detection of ternbutryn. The produced monoclonal antibody has high specificity and sensitivity in the detection of ternbutryn, and the IC 50 value is 2.14 ng / mL. By using the above hybridoma cell line and monoclonal antibody or its composition, a variety of ternbutryn detection products can be developed, such as reagents, test strips, kits, and biochips, etc. These products can meet the ternbutryn detection in different occasions and requirements, providing an efficient, accurate, and convenient means for the specific detection of ternbutryn, and having significant technical effects and wide application values.

[0029] Biological material preservation

[0030] The hybridoma cell line secreting ternbutryn monoclonal antibody is preserved in the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. It is classified and named as monoclonal cell line. The preservation date is October 31, 2024, and the preservation number is CGMCC NO. 46230. Description of the drawings

[0031] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention in combination with the drawings, where

[0032] Figure 1 is the LC-MS / MS characterization diagram of ternbutryn hapten 2 in Example 1 of the present invention;

[0033] Figure 2 is the inhibition standard curve of ternbutryn monoclonal antibody in Example 5 of the present invention. Detailed implementation manners

[0034] The present invention will be 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 be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0035] The culture media involved in the embodiments are as follows:

[0036] RPMI-1640 culture 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, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B12 0.005, sodium bicarbonate 2000.

[0037] The reagents involved in the culture medium are as follows:

[0038] Carbonate buffer solution (CBS): Weigh 1.59 g of Na 2 CO 3 and 2.93 g of NaHCO 3 respectively, dissolve them in a small amount of double-distilled water and then mix. Add double-distilled water to about 800 mL and mix well. Adjust the pH value to 9.6, add double-distilled water to a final volume of 1000 mL, and store at 4°C for later use.

[0039] Phosphate buffer solution (PBS): 8.00 g of NaCl, 0.2 g of KCl, 0.2 g of KH 2 PO 4 , 2.9 g of Na 2 HPO 4 ·12H 2 O, dissolve in 800 mL of pure water, adjust the pH to 7.2 - 7.4 with NaOH or HCl, and make up the volume to 1000 mL;

[0040] PBST: PBS containing 0.05% Tween 20;

[0041] Antibody diluent: PBS containing 0.1% gelatin;

[0042] TMB Chromogenic Solution: Solution A: Na 2 HPO 4 ·12H 2 O 18.43 g, citric acid 9.33 g, made up to 1000 mL with pure water; Solution B: 60 mg of TMB is dissolved in 100 mL of ethylene glycol. Solutions A and B are mixed at a ratio of 5:1 to obtain the TMB chromogenic solution, which is mixed before use.

[0043] The detection methods involved in the examples are as follows:

[0044] Detection method for the inhibition rate of dinoseb: Select the most suitable antigen and antibody concentrations in ic-ELISA through checkerboard experiments. Dilute the antigen to 0.01, 0.03, 0.1, and 0.3 μg / mL with carbonate buffer solution (CBS), and dilute the antibody to 0.03, 0.1, 0.3, and 1 μg / mL with antibody diluent. After selecting the optimal working point, dilute the dinoseb standard to 8 concentrations (0, 0.069, 0.21, 0.62, 1.85, 5.56, 16.7, and 50 ng / mL). According to the operation steps of ic-ELISA, finally plot the graph with OriginPro8.5 (the results are as Figure 1 shown), obtain the dinoseb standard inhibition curve, and calculate IC 50 .

[0045] Example 1: Synthesis of dinoseb hapten

[0046] Since the small molecule of dinoseb does not have immunogenicity and cannot stimulate mice to produce an immune response and then produce antibodies, it is necessary to conjugate dinoseb to a protein through protein conjugation technology to make it acquire immunogenicity; the commonly used reactive groups in protein conjugation technology include amino, carboxyl, hydroxyl, sulfhydryl, etc. In view of the fact that the dinoseb molecular structural formula does not contain these reactive groups, dinoseb is designed for derivation.

[0047] Dinoseb hapten 1 is 3,5-dinitrosalicylic acid (CAS: 609-99-4)

[0048] Derivation of dinoseb hapten 2:

[0049] First, dissolve 2-hydroxy-5-tert-butyl-1,3-dinitrobenzene (1.8 g, 7.6 mmol) in toluene (20 mL), and react with N,N-dimethylformamide (DMF) (5 mL) and thionyl chloride (SOCl 2)Mixing; (1.6 mL, 22.8 mmol). Then stir at 0 °C for 6 h, and obtain a pale yellow solid after rotary evaporation. Wash the precipitate with water and filter to obtain Product A. Then dissolve Product A in tetrahydrofuran (10 mL) and mix it with (iPr)2EtN (1.9 g, 15.2 mmol). Dissolve methyl 4-aminobutyrate (1.3 g, 11.4 mmol) in water, adjust the pH to 8.5, and slowly add the solution of Product A thereto at 0 °C for 6 h. Obtain a yellow solid after rotary evaporation. Finally, dissolve the residue in ethyl acetate (50 mL), and then wash it with HCl (20 mL, 0.1 mol / L) and water (20 mL). Stir the resulting mixture at 75 °C for 7 h, then concentrate to obtain some pale yellow viscous substances. Treat with water and extract with ethyl acetate, then use NaSO 4 Dry, concentrate on a rotary evaporator, and purify on a silica gel column to obtain a pale yellow solid. Dissolve the pale yellow solid in anhydrous methanol (20 mL) at 25 °C, slowly add NaOH (2 M, 20 mL), stir for 10 min and heat it to 45 °C overnight. Filter the mixture through a celite pad, and rinse the solid with ethyl acetate. Concentrate the filtrate under reduced pressure, dissolve the residue in ethyl acetate (100 mL) and wash it successively with water (3×100 mL) and brine (100 mL). Separate the organic layer, dry it with sodium sulfate, filter and concentrate in vacuo to obtain the tebutolol derivative (60 mg, 8%). The LC-MS / MS characterization results of tebutolol hapten 2 are as Figure 1 shown.

[0050] Example 2: Synthesis of tebutolol complete antigen

[0051] Weigh 6 mg of tebutolol hapten 1 and 4.4 mg of N-hydroxysuccinimide (NHS) and dissolve them in 200 μL of N,N-dimethylformamide (DMF), and stir and react at room temperature for 10 min; weigh 7.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), add it to the tebutolol hapten solution, and stir and react at room temperature for 6 - 8 h for activation. Take 6 mg of keyhole limpet hemocyanin (KLH), add it to 3 mL of 0.01 M carbonate buffer solution (CBS), dissolve it thoroughly, and slowly add the activated hapten to the dilution solution in which KLH is dissolved, and stir overnight at room temperature. Then dialyze with 0.01 M PBS solution to remove unreacted small molecules to obtain a relatively pure complete antigen, and identify it by ultraviolet absorption scanning method. The structural formula of tebutolol complete antigen is as follows:

[0052]

[0053] Example 3: Synthesis of tebutolol coating antigen

[0054] Dissolve 3.2 mg of the tebutam hapten 2 and 2.4 mg of N-hydroxysuccinimide (NHS) in 200 μL of anhydrous N,N-dimethylformamide (DMF), and stir the reaction at room temperature for 10 min; dissolve 4.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in the above solution, and stir the reaction at room temperature for 6 - 8 h to obtain the 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 solution, and stir overnight at room temperature. Then, dialyze the reaction solution with 0.01 M PBS solution to remove unreacted small molecules to obtain the coating antigen. The structural formula of the tebutam coating antigen is shown below:

[0055]

[0056] Example 4: Preparation of a hybridoma cell line secreting tebutam monoclonal antibody

[0057] 1. Obtaining animal immunity

[0058] After emulsifying the tebutam complete antigen with an equal amount of Freund's adjuvant, perform multiple subcutaneous injections on the back of the neck of BALB / c mice for immunization (except for the booster immunization); use complete Freund's adjuvant for the first immunization, with a dose of 100 μg / mouse; use incomplete Freund's adjuvant for multiple booster immunizations and halve the dose to 50 μg / mouse; for the booster immunization, do not use adjuvant, directly dilute with physiological saline and inject intraperitoneally, and halve the dose again to 25 μg / mouse; the interval between the first immunization and the second booster immunization is one month, the interval between multiple booster immunizations is 21 days, and the interval between the booster immunization and the last booster immunization is 18 - 21 days; observe the immune effect of the mice by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), that is, detect the titer and inhibition of the mouse serum, and screen out the mice with high sensitivity of tebutam antibody in the serum.

[0059] 2. Cell fusion

[0060] Three days after the booster immunization, perform cell fusion according to the conventional PEG (polyethylene glycol, molecular weight 4000) method. The specific steps are as follows:

[0061] a. Take blood from the tail vein, sacrifice the mouse by cervical dislocation, immediately put it into 75% alcohol for disinfection, soak for about 5 min, aseptically take out the spleen of the mouse, moderately grind it with the rubber head of a syringe and obtain a spleen cell suspension through a 200-mesh cell sieve, collect it, centrifuge (1200 rpm, 8 min), wash the spleen cells three times with RPMI-1640 medium, and after the last centrifugation, dilute the spleen cells to a certain volume, count them, and set aside;

[0062] b. Collect SP2 / 0 cells: 7 - 10 days before fusion, culture SP2 / 0 tumor cells in RPMI - 1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO 2 incubator for amplification. Before fusion, the number of SP2 / 0 tumor cells is required to reach 1 - 4×10 7 . Ensure that the SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. At the time of fusion, collect the tumor cells, suspend them in RPMI - 1640 basal culture medium, and perform cell counting;

[0063] c. 7 - minute fusion process: At the 1st minute, slowly add 1 mL of PEG 4000 to the cells at an increasing rate; at the 2nd minute, let it stand; at the 3rd and 4th minutes, add 1 mL of RPMI - 1640 medium dropwise within 1 minute; at the 5th and 6th minutes, add 2 mL of RPMI - 1640 medium dropwise within 1 minute; at the 7th minute, add 1 mL of RPMI - 1640 medium every 10 seconds. Shake the solution continuously at other times except the 2nd minute. Then incubate at 37°C for 5 minutes; centrifuge (800 rpm, 8 minutes), discard the supernatant, resuspend in RPMI - 1640 screening culture medium containing 20% fetal bovine serum and 2% of 50×HAT, and add it to a 96 - well cell plate at 200 μL / well, and place it in a 37°C, 5% CO 2 incubator for culture.

[0064] 3. Cell screening and cell line establishment

[0065] On the 3rd day after cell fusion, perform a semi - medium change of the fusion cells with RPMI - 1640 screening culture medium, and on the 5th day, perform a full - medium change with RPMI - 1640 transition culture medium containing 20% fetal bovine serum and 1% of 100×HT. On the 7th day, take the cell supernatant for screening.

[0066] The screening is carried out in two steps: First, use the ic - ELISA method to screen out positive cell wells, and second, use tebutolol as a standard product and use the ic - ELISA method to measure the inhibitory effect on positive cells.

[0067] Select the cell wells with good inhibition of the tebutolol standard product, and perform sub - cloning by the limiting dilution method. After seven days, detect them by the same method.

[0068] Perform at least three sub - clonings according to the above method to finally obtain the tebutolol monoclonal antibody cell line.

[0069] Example 5: Preparation and identification of tebutolol monoclonal antibody

[0070] Take 8 - 10 - week - old BALB / c mice, and inject 1 mL of sterile paraffin oil intraperitoneally into each mouse; 7 days later, inject 1×106 For the tebutamol hybridoma cells, ascites was collected starting from the seventh day, and the ascites was subjected to antibody purification by the octanoic acid-ammonium sulfate saturation method.

[0071] Under mildly acidic conditions, n-octanoic acid can precipitate other miscellaneous proteins in the ascites except for IgG immunoglobulins, followed by centrifugation to discard the precipitate; then, an ammonium sulfate solution with an equal saturation is used to precipitate the monoclonal antibody of the IgG type, followed by centrifugation to discard the supernatant. After dissolution with a 0.01 M PBS solution (pH = 7.4), dialysis is performed to remove salts, and finally, the purified monoclonal antibody is stored at -20 °C.

[0072] Using indirect competitive ELISA, the standard inhibition curve of the tebutamol monoclonal antibody was measured. As Figure 2 shown, tebutamol has good detection sensitivity (IC 50 values are 2.14 ng / mL respectively), and it can be used for the immunoassay detection of tebutamol.

[0073] Cross-reaction experiments were conducted with tebutamol structural analogs (pentachloronitrobenzene, dinobuton, butralin, trifluralin, flumetralin). The experimental results were as follows: there were no significant differences in the absorbance values of each well of the enzyme-labeled plate for diclofop-methyl with the change of the standard product dilution concentration. The specific results are shown in Table 1. This result indicates that the cross-reaction rate of the monoclonal antibody with other structural analogs is very low, and it has good sensitivity to tebutamol. Among them, the cross-reaction rate (%) = (tebutamol IC 50 ) / (analog IC 50 ) × 100%.

[0074] Table 1 Cross-reaction rate of tebutamol monoclonal antibody with tebutamol structural analogs

[0075]

[0076] Example 6: Application of tebutamol monoclonal antibody

[0077] The monoclonal antibody prepared from the hybridoma cell line through in vivo ascites was applied to the ELISA spiking recovery test of tebutamol. The specific steps are as follows:

[0078] (1) The coating antigen diluted with carbonate buffer solution (CBS) at a concentration of 0.1 μg / mL was used to coat a 96-well enzyme-labeled plate, 100 μL per well. After baking at 37 °C for 2 h, the plate was washed three times with PBST washing solution, 200 μL per well each time, for 3 min each time, and then patted dry;

[0079] (2) Blocking was performed with CBS containing 0.2% gelatin, 200 μL per well. After baking at 37 °C for 2 h, the plate was washed three times with PBST washing solution, 200 μL per well each time, for 3 min each time, and then patted dry;

[0080] (3) Prepare standard solutions of ternbutol at 0, 0.037, 0.111, 0.333, 1, 3, 9, and 27 ng / mL respectively using phosphate buffer solution (PBS). Add the standard solutions and the extract of the sample to be detected into the well plate that has been blocked, 50 μL per well. Repeat each sample in 3 wells. Then add 50 μL of the ternbutol monoclonal antibody diluted to 0.1 μg / mL to each well. After reacting at 37 °C for 30 min, wash the plate and pat it dry;

[0081] (4) Add 100 μL of the secondary antibody, HRP-labeled goat anti-mouse IgG, diluted 1:3000 with PBS containing 0.1% gelatin to each well. After reacting at 37 °C for 30 min, wash the plate and pat it dry;

[0082] (5) Add 100 μL of the TMB chromogenic solution to each well. After developing color at 37 °C for 15 min, add 50 μL of 2M H 2 SO 4 termination solution, and measure the absorbance at 450 nm;

[0083] (6) Spike recovery and sample pretreatment:

[0084] Select rice as the test sample. The sample to be tested is crushed and passed through a 20-mesh standard sieve. Weigh three portions of the sample, 20 g each. Add ternbutol standard products at 0.5 ppb, 2 ppb, and 5 ppb respectively to the samples (set the spiked concentration according to the linear range of the antibody and IC 50 . Add 10 mL of water, vortex and mix well, let stand for 30 min, and filter by suction. Add 50 mL of acetone to the sample, shake on an electric shaker for 30 min, filter through a fast qualitative filter paper into a beaker. Extract the residue with 30 mL of acetone again according to the above method, wash the residue with 30 mL of acetone twice, combine the washing solutions into the beaker, concentrate to nearly dry at 50 °C in a water bath, and redissolve with 5 mL of 10% acetone PBS solution (i.e., dilute five times to reduce the influence of the sample matrix).

[0085] The spike recovery test was carried out by indirect competitive ELISA, and the recovery rates were 89.34 - 112.4% for ternbutol.

[0086] Obviously, the above embodiments are only examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A hybridoma cell line secreting a monoclonal antibody to Telofol, characterized in that: The deposit number of the hybridoma cell line is CGMCC NO.46230.

2. Use of the hybridoma cell line according to claim 1 in the preparation of a monoclonal antibody against Teleb.

3. A monoclonal antibody to Telofol, characterized in that: The telofib 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 Telebol monoclonal antibody according to claim 3 or the composition according to claim 4 in preparing a Telebol detection product.

6. The use according to claim 5, characterized in that: The telofob detection product includes reagents, test strips, test kits or biochips.

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

8. A kit, characterized in that: The kit contains the hybridoma cell line according to claim 1, the telomerase 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 telofol-coated antigen, a telofol 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 of Telebol according to claim 3, the composition according to claim 4, the test strip according to claim 7 or the kit according to claim 8 in detecting Telebol.

Images