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

Abstract

The invention relates to a hybridoma cell strain secreting a methylphenate monoclonal antibody and application of the hybridoma cell strain. The hybridoma cell strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on October 31, 2024, the preservation number is CGMCC No.46223, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the hybridoma cell strain is classified and named as a monoclonal cell strain. The methyl piperaacetate monoclonal antibody secreted by the hybridoma cell strain disclosed by the invention has better detection sensitivity (IC50 value is 1.02 ng / mL) to methyl piperaacetate, and can be used for residue detection of methyl piperaacetate.

Description

Technical Field

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

[0002] The common name of methylphenidate in English is Methylphenidate, which is a central nervous system stimulant drug, mainly used to treat children's attention deficit hyperactivity disorder, narcolepsy, etc. It can improve attention, let children concentrate, and improve learning ability to a certain extent. However, long-term use is addictive and belongs to a class of psychotropic drugs strictly controlled by the state. Therefore, it can only be dispensed in hospitals with a doctor's prescription and is limited in supply according to the patient's pathological and physiological conditions and treatment courses. However, some institutions, parents or illegal elements take advantage of the drug properties of methylphenidate to illegally obtain methylphenidate, which greatly threatens the social legal security. Therefore, it is very important to conduct high-sensitivity detection of methylphenidate.

[0003] At present, the detection method of methylphenidate is mainly instrumental detection, and gas chromatography, liquid chromatography and gas chromatography-mass spectrometry are commonly used. 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, a rapid and simple method for detecting methylphenidate residues is needed.

[0004] Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method. During the detection, the sample pre-treatment 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 on-site rapid detection of a large number of samples, so it has been widely used in drug residue analysis. The premise of using ELISA to detect methylphenidate is to obtain a monoclonal antibody with high specificity and high sensitivity to methylphenidate. Therefore, it is very important to find a method for preparing a monoclonal antibody with high specificity and high sensitivity to methylphenidate. The inventor tried to prepare methylphenidate monoclonal antibodies through hybridoma cells, but in the process of preparing hybridoma cell lines that can secrete methylphenidate monoclonal antibodies, how to prepare methylphenidate haptens and complete antigens and how to make mice produce strong immune effects still need further research; how to make the prepared hybridoma cell lines successfully secrete methylphenidate monoclonal antibodies still need further research; how to make the secreted methylphenidate monoclonal antibodies have strong specificity and high sensitivity also need further research. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a hybridoma cell line secreting a monoclonal antibody against methylphenidate and its application. The monoclonal antibody against methylphenidate secreted by the hybridoma cell line has a good detection sensitivity (IC 50 The value is 1.02ng / mL), which can be used to establish an immunological detection method for methylphenidate and detect the residues of methylphenidate in urine.

[0006] The technical solution of the present invention is as follows:

[0007] The first object of the present invention is to provide a hybridoma cell line that secretes methylphenidate monoclonal antibodies. The hybridoma cell line was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on October 31, 2024, with a deposit number of CGMCC No.46223, a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and was classified and named a monoclonal cell line.

[0008] The second object of the present invention is to provide a methylphenidate monoclonal antibody secreted and produced by the hybridoma cell line.

[0009] The third object of the present invention is to provide a methylphenidate hapten for preparing the hybridoma cell line, wherein the structural formula of the methylphenidate hapten is:

[0010]

[0011] The fourth object of the present invention is to provide a composition comprising the hybridoma cell line and / or the methylphenidate monoclonal antibody.

[0012] A fifth object of the present invention is to provide a kit for detecting methylphenidate, wherein the kit contains at least one of the hybridoma cell line, the methylphenidate monoclonal antibody and the composition.

[0013] In one embodiment of the present invention, the kit is selected from an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

[0014] A sixth object of the present invention is to provide a test strip for detecting methylphenidate, wherein the test strip contains at least one of the hybridoma cell line, the methylphenidate monoclonal antibody and the composition.

[0015] In one embodiment of the present invention, the test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane and a water absorbent pad, the nitrocellulose membrane is provided with a detection line and a quality control line in sequence, and the conjugate pad is coated with the methylphenidate monoclonal antibody.

[0016] A seventh object of the present invention is to provide a chip for detecting methylphenidate, wherein the chip contains at least one of the hybridoma cell line, the methylphenidate monoclonal antibody and the composition.

[0017] The eighth object of the present invention is to provide use of the hybridoma cell line, the methylphenidate monoclonal antibody, the composition, the kit, the test strip or the chip in detecting methylphenidate.

[0018] The present invention provides a method for preparing the hybridoma cell line secreting methylphenidate monoclonal antibody, comprising the following steps:

[0019] Step 1: preparing methylphenidate hapten and methylphenidate complete antigen, and emulsifying the obtained methylphenidate complete antigen with Freund's adjuvant or incomplete Freund's adjuvant to prepare an immunogen;

[0020] Step 2: The obtained immunogen is injected subcutaneously into the back of BALB / c mice for multiple immunizations, with complete Freund's adjuvant used for the first immunization and incomplete Freund's adjuvant used for the booster immunization;

[0021] Step 3: Collect blood from the mice that have undergone the above immunization process, and detect the immune titer and immunosuppression ability of the mouse serum by indirect ELISA to screen out mice with high sensitivity of methylphenidate antibodies in the serum;

[0022] Step 4: The screened mice are immunized by intraperitoneal injection, and the immunization is performed with complete methylphenidate antigen without Freund's adjuvant;

[0023] Step 5: The spleen cells of BALB / c mice after sprint immunization were fused with myeloma cells, the fused cells were screened and cultured with HAT medium, the positive cell wells were detected by indirect ELISA, and the inhibition effect of the positive cell wells was further determined by indirect competitive ELISA. The positive cell wells with the best inhibition were subcloned by limiting dilution method, and finally the hybridoma cell line that could secrete highly sensitive methylphenidate monoclonal antibodies was screened out;

[0024] In one embodiment of the present invention, in step 1, the molecular formula of the methylphenidate hapten is as follows:

[0025]

[0026] In one embodiment of the present invention, in step 1, the molecular formula of the methylphenidate complete antigen is as follows:

[0027]

[0028] In one embodiment of the present invention, the preparation method of the complete methylphenidate antigen is as follows: dissolving the methylphenidate hapten, N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in anhydrous N,N-dimethylformamide (DMF), stirring for reaction, and obtaining an activated methylphenidate hapten solution, i.e., solution A; diluting keyhole limpet hemocyanin with carbonate buffer solution (CBS) to obtain solution B; slowly adding solution A to solution B for reaction to obtain a reaction solution; and dialyzing the reaction solution with phosphate buffered saline (PBS) to obtain a complete antigen.

[0029] In one embodiment of the present invention, in steps 2 and 4, the interval between the first immunization and the booster immunization is one month, the interval between the booster immunizations is 21 days, and the interval between the booster immunization and the sprint immunization is 18 to 21 days.

[0030] In one embodiment of the present invention, in steps 2 and 4, the first immunization dose is 20 μg / mouse to 100 μg / mouse, the booster immunization dose is 10 μg / mouse to 50 μg / mouse, and the sprint immunization dose is 5 μg / mouse to 25 μg / mouse. Preferably, the first immunization dose is 100 μg / mouse, the booster immunization dose is 50 μg / mouse, and the sprint immunization dose is 25 μg / mouse.

[0031] In one embodiment of the present invention, in steps 2 and 4, the immunization process includes 1 initial immunization, 4 booster immunizations and 1 sprint immunization;

[0032] In one embodiment of the present invention, in step 3, the blood is collected on the 7th day after the end of the third immunization process.

[0033] In one embodiment of the present invention, in step 5, the cell fusion is performed 3 days after the end of the sprint immunization.

[0034] In one embodiment of the present invention, in step 5, the cell fusion is performed by polyethylene glycol (PEG4000) method.

[0035] In one embodiment of the present invention, in step 5, the culture medium is RPMI-1640 culture medium.

[0036] In one embodiment of the present invention, in step 5, the number of subcloning is 2 to 5 times, preferably 4 times.

[0037] The present invention also provides application of the method for preparing the hybridoma cell line secreting the methylphenidate monoclonal antibody in preparing the methylphenidate monoclonal antibody.

[0038] The invention provides a method for preparing the methylphenidate monoclonal antibody, which comprises the following steps: taking a BALB / c mouse, intraperitoneally injecting paraffin oil, and then intraperitoneally injecting a hybridoma cell line with a preservation number of CGMCC No.46223, collecting ascites after the injection, purifying the ascites, and cryopreserving the obtained monoclonal antibody.

[0039] The present invention provides application of the preparation method of the methylphenidate monoclonal antibody in identifying methylphenidate.

[0040] The above technical solution of the present invention has the following advantages compared with the prior art:

[0041] (1) The methylphenidate monoclonal antibody obtained by the present invention has a good detection sensitivity (IC 50 value is 1.02ng / mL);

[0042] (2) The methylphenidate monoclonal antibody cell line obtained in the present invention can be used for immunoassay detection.

[0043] Biomaterial Deposit

[0044] A hybridoma cell line MOD that secretes methylphenidate monoclonal antibodies has been deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named as a monoclonal cell line. The deposit date is October 31, 2024, and the deposit number is CGMCC No.46223. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] 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:

[0046] Figure 1 This is the inhibition standard curve of the methylphenidate monoclonal antibody of the present invention on methylphenidate. DETAILED DESCRIPTION

[0047] 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.

[0048] The culture medium involved in the following examples is as follows:

[0049] 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. Nicotinamide 1. Choline chloride 3. Pyridoxine hydrochloride 1. Riboflavin 0.2. Thiamine hydrochloride 1. Vitamin B12 0.005. Sodium bicarbonate 2000.

[0050] The reagents involved in the following examples are as follows:

[0051] Carbonate buffer (CBS): Weigh 1.59 g Na2CO3 and 2.93 g 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.

[0052] Phosphate buffered saline (PBS): 8.00 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;

[0053] PBST: PBS containing 0.05% Tween 20;

[0054] Antibody diluent: PBS containing 0.1% gelatin;

[0055] 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. A and B solutions are mixed in a ratio of 5:1 to form TMB colorimetric solution, which needs to be mixed again before use.

[0056] The detection methods involved in the following embodiments are as follows:

[0057] Methylphenidate inhibition rate detection method: The most appropriate antigen and antibody concentrations in ic-ELISA were selected by chessboard test. The antigen was diluted to 0.01μg / mL, 0.03μg / mL, 0.1μg / mL and 0.3μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.03μg / mL, 0.1μg / mL, 0.3μg / mL and 1μg / mL with antibody diluent. After selecting the optimal working point, the methylphenidate standard was diluted to 8 concentrations (0, 0.3ng / mL, 1ng / mL, 3ng / mL, 9ng / mL, 27ng / mL and 81ng / mL), and the ic-ELISA operation steps were followed. Finally, OriginPro 8.5 was used to draw the graph (the results are shown in Figure 2). Figure 1 As shown), obtain the standard inhibition curve of methylphenidate and calculate IC 50 .

[0058] Example 1: Synthesis of Methylphenidate Hapten

[0059] Since the small molecule of methylphenidate is not immunogenic and cannot stimulate mice to produce an immune response and then produce antibodies, methylphenidate needs to be coupled to the protein through protein coupling technology to make it immunogenic; the active groups commonly used in protein coupling technology include amino, carboxyl, hydroxyl, thiol, etc. Since the molecular structure of methylphenidate does not contain these active groups, methylphenidate is derivatized.

[0060] 5-(tert-butylcarbonyl)aminopentanoic acid (4 g, 0.0183 mol) was added to a THF (54 mL) solution, CDI (3.26 g, 0.02013 mol) was added, and the mixture was stirred at 15 ° C for 1 hour (solution a). 2-(4-bromophenyl)acetic acid methyl ester (12.58 g, 0.0549 mol) was dissolved in THF (90 mL), and LDA (2M, 27.4 mL, 0.0549 mol) was added dropwise at -40 ° C. After addition, the resulting mixture was stirred at the same temperature for 1.5 hours, and the reaction was quenched with a saturated aqueous ammonium chloride solution, and 1.0 M HCl was added to adjust the pH to 4-5. The mixture was extracted with ethyl acetate, and the combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by silica gel flash column to obtain intermediate product 1 as a yellow oil (7.6 g, yield: 92%).

[0061] TFA (10.09 g, 0.0885 mol) was added to a DCM (35 mL) solution of intermediate 1. The reaction mixture was stirred at 15 ° C for 16 hours. The reaction mixture was concentrated and the residue was redissolved in DCM (100 mL). It was separated after adjusting to pH = 10 with sodium sulfate (Sat.Na2CO3). The aqueous phase was extracted with DCM (100 mL). The combined DCM was washed with brine (50 mL), dried and concentrated. The residue was fermented with PE (20 mL), filtered, washed with PE (10 mL), and dried to give intermediate 2 (3.68 g, yield: 64%) as a yellow solid.

[0062] The intermediate product 2, benzyl-4-vinyl ester (1.45 g, 0.00768 mol), bis(triphenylphosphine)palladium chloride (0.45 g, 0.00064 mol), copper iodide (0.24 g, 0.00128 mol) and TEA (3.24 g, 0.032 mol) were stirred in DMF (20 mL) at 80 ° C under N2 atmosphere for 16 h. The mixture was diluted with H2O (100 mL), extracted with EA (100 mL), washed with brine (60 mL) in combination with EA, and dried and concentrated. The residue was purified by silica gel flash column (EA in PE was 15%) to obtain the intermediate product 3 as a yellow solid (1.21 g, yield 41%).

[0063] Palladium / carbon (116 mg) and Pd(OH)2 / C (118 mg) were added to a THF (2 ml) / MeOH (8 mL) solution and mixed with the intermediate product 3 (350 mg, 0.8383 mmol). The reaction mixture was stirred at 60°C under H2 atmosphere for 16 hours. The reaction mixture was filtered to remove the catalyst, and the filtrate was concentrated and dried to obtain a crude oil. The residue was purified by Prep-HPLC (NH4HCO3 as an additive) to obtain the final product as a white solid (70 mg, yield: 25%), i.e., methylphenidate hapten.

[0064] Example 2: Synthesis of complete methylphenidate antigen

[0065] Weigh 6 mg of methylphenidate hapten and 4.4 mg of N-hydroxysuccinimide (NHS), dissolve in 200 μL of N,N-dimethylformamide (DMF), and stir at room temperature for 10 minutes; then weigh 7.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), add to the methylphenidate hapten solution, stir at room temperature for 6-8 hours to activate. Take 6 mg of keyhole limpet hemocyanin (KLH), add to 3 mL of 0.01 M carbonate buffer (CBS), fully dissolve, slowly add the activated hapten to the diluent in which KLH is dissolved, and stir at room temperature overnight. Then dialyze with 0.01 M PBS solution to remove unreacted small molecules, obtain a relatively pure complete antigen, and identify it by ultraviolet absorption scanning method.

[0066] Example 3: Synthesis of Methylphenidate Coating

[0067] 3.2 mg of methylphenidate hapten and 2.4 mg of N-hydroxysuccinimide (NHS) were dissolved in 200 μL of anhydrous N,N-dimethylformamide (DMF) and stirred at room temperature for 10 min; 4.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in the above solution and stirred at room temperature for 6-8 h to obtain a hapten activation solution; 6 mg of chicken ovalbumin (OVA) was dissolved in carbonate buffer (CBS); the hapten activation solution was slowly added to the protein diluent and stirred at room temperature overnight. Then, the reaction solution was dialyzed with 0.01 M PBS solution to remove unreacted small molecules to obtain a coating source.

[0068] Example 4: Preparation of hybridoma cell lines secreting methylphenidate monoclonal antibodies

[0069] 1. Acquisition of animal immunity

[0070] After the complete methylphenidate antigen was mixed and emulsified with an equal amount of Freund's adjuvant, BALB / c mice were immunized by multiple subcutaneous injections at the back of the neck (except for sprint immunization); the first immunization used complete Freund's adjuvant, with a dose of 100 μg / mouse; multiple booster immunizations used incomplete Freund's adjuvant and the dose was halved to 50 μg / mouse; no adjuvant was used for sprint immunization, and it was directly diluted with normal saline and injected intraperitoneally, and the dose was halved again to 25 μg / mouse; the first immunization and the second booster immunization were separated by one month, the multiple booster immunizations were separated by 21 days, and the sprint immunization and the last booster immunization were separated by 18-21 days; the immune effect of mice was observed by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), that is, the titer and inhibition of mouse serum were detected;

[0071] 2. Cell fusion

[0072] Three days after the sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 4000) method. The specific steps are as follows:

[0073] a. Cut off the tail to collect blood. After killing the mice by cervical dislocation, immediately put them in 75% alcohol for disinfection and soak for about 5 minutes. Take out the spleen of the mice aseptically, grind them moderately with the rubber tip of the syringe and pass them through a 200-mesh cell sieve to obtain a spleen cell suspension, collect them, centrifuge them (1200rpm, 8min), wash the spleen cells three times with RPMI-1640 medium, dilute the spleen cells to a certain volume after the last centrifugation, count them, and set aside;

[0074] b. Collect SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were amplified in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. Before fusion, the number of SP2 / 0 tumor cells was required to reach 1-4×10 7 , 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;

[0075] c. Fusion process 7min: In the first minute, 1mL of PEG 4000 was added to the cells from slow to fast; in the second minute, it was allowed to stand; in the third and fourth minutes, 1mL of RPMI-1640 medium was added within 1min; in the fifth and sixth minutes, 2mL of RPMI-1640 medium was added within 1min; in the seventh minute, 1mL of RPMI-1640 medium was added every 10s. Except for the second minute, the solution was shaken continuously at other times. Then, it was warmed at 37℃ for 5min; centrifuged (800rpm, 8min), the supernatant was discarded, and the cell was resuspended in RPMI-1640 screening culture medium containing 20% ​​fetal bovine serum and 2% 50×HAT, and added to 96-well cell plates at 200μL / well, and cultured in a 37℃, 5% CO2 incubator.

[0076] 3. Cell screening and cell line establishment

[0077] On the third day after cell fusion, the fused cells were half-changed with RPMI-1640 screening medium, and on the fifth day, the medium was fully replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On the seventh day, the cell supernatant was taken for screening.

[0078] The screening was divided into two steps: the first step was to screen out the positive cell wells using the ic-ELISA method, and the second step was to use methylphenidate as a standard and use the ic-ELISA method to measure the inhibitory effect on the positive cells.

[0079] The cell wells with better inhibition of methylphenidate standard were selected, subcloned by limiting dilution method, and tested by the same method seven days later.

[0080] Subcloning was performed at least three times according to the above method to finally obtain a methylphenidate monoclonal antibody cell line.

[0081] Example 5: Preparation and identification of monoclonal antibodies to methylphenidate

[0082] 8-10 week old BALB / c mice were taken and each mouse was intraperitoneally injected with 1 mL of sterile paraffin oil. Seven days later, each mouse was intraperitoneally injected with 1×10 6 Methylphenidate hybridoma cells, ascites was collected from the seventh day and the antibody was purified by the caprylic acid-saturated ammonium sulfate method.

[0083] Under slightly acidic conditions, octanoic acid can precipitate other impurities in the ascites except IgG immunoglobulin, 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°C.

[0084] IC of methylphenidate monoclonal antibody was measured using indirect competitive ELISA 50 The value was 1.02 ng / mL, indicating good sensitivity to methylphenidate and can be used for methylphenidate immunoassay detection.

[0085] Example 6: Application of methylphenidate monoclonal antibody

[0086] The monoclonal antibody prepared by hybridoma cell line through ascites in vivo was applied to ELISA spike recovery test of methylphenidate. The specific steps are as follows:

[0087] (1) Coat a 96-well ELISA plate with 0.1 μg / mL of the coating agent diluted with carbonate buffer (CBS), 100 μL per well, bake at 37°C for 2 h, wash the plate three times with PBST, 200 μL per well each time, 3 min each time, and pat dry;

[0088] (2) Block with CBS containing 0.2% gelatin, 200 μL per well, bake at 37°C for 2 h, wash the plate three times with PBST, 200 μL per well each time, 3 min each time, and pat dry;

[0089] (3) Prepare 0, 0.3 ng / mL, 1 ng / mL, 3 ng / mL, 9 ng / mL, 27 ng / mL and 81 ng / mL of methylphenidate standard solutions using phosphate buffered saline (PBS), add the standard solutions and the sample extracts to be tested to the blocked ELISA plate, 50 μL per well, repeat 3 wells for each sample, then add 50 μL of methylphenidate monoclonal antibody diluted to 0.1 μg / mL to each well, react at 37°C for 30 min, wash the plate and pat dry;

[0090] (4) Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 in PBS containing 0.1% gelatin to each well, react at 37°C for 30 min, wash the plate and pat dry;

[0091] (5) Add 100 μL of TMB colorimetric solution to each well, develop at 37°C for 15 min, then add 50 μL of 2 M H2SO4 stop solution to each well and measure the absorbance at 450 nm;

[0092] (6) Addition recovery and sample pretreatment:

[0093] Urine was selected as the test sample.

[0094] Add 0.1 ppb, 1 ppb, and 10 ppb of methylphenidate standard to the samples (according to the linear range of the antibody and IC 50 Set the concentration of addition) and shake on an electric shaker to mix. The recovery rate of addition recovery test was 106%, 93% and 114% respectively by indirect competitive ELISA.

[0095] 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 against methylphenidate, characterized in that: The hybridoma cell line was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 31, 2024, with the deposit number CGMCC No.46223, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named as a monoclonal cell line.

2. A methylphenidate monoclonal antibody, characterized in that: The hybridoma cell line according to claim 1 is secreted and produced.

3. A methylphenidate hapten for preparing the hybridoma cell line according to claim 1, characterized in that: The structural formula of the methylphenidate hapten is:

4. A composition, characterized in that The composition contains the hybridoma cell line according to claim 1 and / or the methylphenidate monoclonal antibody according to claim 2.

5. A kit for detecting methylphenidate, characterized in that: The kit contains at least one of the hybridoma cell line according to claim 1, the methylphenidate monoclonal antibody according to claim 2, and the composition according to claim 4.

6. The kit according to claim 5, characterized in that The kit is selected from an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

7. A test strip for detecting methylphenidate, characterized in that: The test strip contains at least one of the hybridoma cell line according to claim 1, the methylphenidate monoclonal antibody according to claim 2, and the composition according to claim 4.

8. The test strip according to claim 7, characterized in that: The test strip comprises a sample pad, a conjugation pad, a nitrocellulose membrane and a water absorbent pad, wherein a detection line and a quality control line are arranged in sequence on the nitrocellulose membrane, and the conjugation pad is coated with the methylphenidate monoclonal antibody according to claim 2.

9. A chip for detecting methylphenidate, characterized in that: The chip contains at least one of the hybridoma cell line according to claim 1, the methylphenidate monoclonal antibody according to claim 2, and the composition according to claim 4.

10. Use of the hybridoma cell line according to claim 1, the methylphenidate monoclonal antibody according to claim 2, the composition according to claim 4, the kit according to claims 5-6, the test strip according to claims 6-7 or the chip according to claim 9 in detecting methylphenidate.

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