Hybridoma cell strain secreting p-methylphenethyl acetate monoclonal antibody and application thereof

Abstract

This invention relates to a hybridoma cell line that secretes a monoclonal antibody against methylphenidate and its application. The hybridoma cell line was deposited on October 31, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46223, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and is classified as a monoclonal cell line. The monoclonal antibody against methylphenidate secreted by this hybridoma cell line exhibits good detection sensitivity (IC50) for methylphenidate. 50 With a value of 1.02 ng / mL, it can be used for the detection of methylphenidate residues.

Description

A hybridoma cell line secreting methylphenidate monoclonal antibody and its application Technical Field

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

[0002] Methylphenidate, also known as central nervous system stimulant, is primarily used to treat attention deficit hyperactivity disorder (ADHD) and narcolepsy in children. It can improve attention, enhance concentration, and to some extent, improve learning ability. However, long-term use is addictive and it is a strictly controlled psychotropic drug. Therefore, it can only be dispensed in hospitals with a doctor's prescription, and the supply is limited according to the patient's pathophysiological condition and treatment duration. However, some institutions, parents, or criminals exploit the drug's properties to illegally obtain methylphenidate, posing a significant threat to public safety and the rule of law. Therefore, highly sensitive detection of methylphenidate is crucial.

[0003] Currently, the main methods for detecting methylphenidate are instrumental, with gas chromatography, liquid chromatography, and gas chromatography-mass spectrometry being commonly used. While these chromatographic methods offer high sensitivity and specificity, they also have 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 methylphenidate residues is needed.

[0004] Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method. It requires simple sample pretreatment, involves few purification steps, has a large analytical capacity, low detection cost, and is easy to operate, making it suitable for rapid on-site detection of large numbers of samples. Therefore, it is widely used in drug residue analysis. However, the prerequisite for using ELISA to detect methylphenidate is obtaining a monoclonal antibody with high specificity and sensitivity to methylphenidate. Therefore, finding a method to prepare a monoclonal antibody with high specificity and sensitivity to methylphenidate is crucial. The inventors attempted to prepare methylphenidate monoclonal antibodies using hybridoma cells. However, further research is needed on how to prepare methylphenidate hapten and complete antigen, how to induce a strong immune response in mice, how to ensure the successful secretion of methylphenidate monoclonal antibodies by the prepared hybridoma cell lines, and how to ensure that the secreted methylphenidate monoclonal antibodies have high specificity and sensitivity. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes a monoclonal antibody against methylphenidate and its applications. The monoclonal antibody against methylphenidate secreted by this hybridoma cell line exhibits good detection sensitivity (IC50) for methylphenidate. 50 With a value of 1.02 ng / mL, it can be used to establish an immunological detection method for methylphenidate to detect methylphenidate residues in urine.

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

[0007] The first objective of this invention is to provide a hybridoma cell line that secretes methylphenidate monoclonal antibody. This hybridoma cell line was deposited on October 31, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46223, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and is classified as a monoclonal cell line.

[0008] A second objective of this invention is to provide a methylphenidate monoclonal antibody secreted by the hybridoma cell line described above.

[0009] A third objective of this invention is to provide a methylphenidate hapten for preparing the aforementioned hybridoma cell line, wherein the structural formula of the methylphenidate hapten is:

[0010]

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

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

[0013] In one embodiment of the present invention, the kit is selected from enzyme-linked immunosorbent assay (ELISA) kits, fluorescence immunoassay kits, or chemiluminescent immunoassay kits.

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

[0015] In one embodiment of the present invention, the test strip includes a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane is provided with a detection line and a 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, the chip containing at least one of the hybridoma cell line, the methylphenidate monoclonal antibody, and the composition.

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

[0018] This invention provides a method for preparing a hybridoma cell line that secretes methylphenidate monoclonal antibody, comprising the following steps:

[0019] Step 1: Prepare methylphenidate hapten and methylphenidate complete antigen. Emulsify the obtained methylphenidate complete antigen with Freund's adjuvant or incomplete Freund's adjuvant to prepare an immunogen.

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

[0021] Step 3: Collect blood from mice that have undergone the above immunization process, and detect the serum immune titer and immunosuppressive capacity of mice by indirect ELISA to screen out mice with high sensitivity of methylphenidate antibody in serum.

[0022] Step 4: The selected mice were immunized by intraperitoneal injection using methylphenidate complete antigen without Freund's adjuvant.

[0023] Step 5: Fuse spleen cells and myeloma cells from BALB / c mice after sprint immunization. The fused cells are cultured in HAT medium and screened. Positive cell pores are detected by indirect ELISA. The inhibitory effect of positive cell pores is further determined by indirect competitive ELISA. The positive cell pores with the best inhibition are subcloned by limiting dilution method. Finally, hybridoma cell lines that can secrete highly sensitive methylphenidate monoclonal antibodies are 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 method for preparing the methylphenidate complete antigen is as follows: methylphenidate hapten, N-hydroxysuccinimide (NHS), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) are dissolved in anhydrous N,N-dimethylformamide (DMF), and the mixture is stirred to obtain an activated methylphenidate hapten solution, i.e., solution A; keyhole hemocyanin is diluted with carbonate buffer solution (CBS) to obtain solution B; solution A is slowly added to solution B to carry out the reaction to obtain a reaction solution; the reaction solution is dialyzed with phosphate buffered saline (PBS) to obtain the complete antigen.

[0029] In one embodiment of the present invention, in steps 2 and 4, the interval between the initial immunization and the booster immunization is one month, the interval between booster immunizations is 21 days, and the interval between 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 initial immunization dose is 20 μg / animal to 100 μg / animal, the booster immunization dose is 10 μg / animal to 50 μg / animal, and the final immunization dose is 5 μg / animal to 25 μg / animal. Preferably, the initial immunization dose is 100 μg / animal, the booster immunization dose is 50 μg / animal, and the final immunization dose is 25 μg / animal.

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

[0032] In one embodiment of the present invention, in step 3, the blood collection is performed on the 7th day after the completion of the 3rd 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 the 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 operations is 2 to 5 times, preferably 4 times.

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

[0038] The present invention provides a method for preparing the methylphenidate monoclonal antibody as follows: BALB / c mice are injected intraperitoneally with paraffin oil, followed by intraperitoneal injection of a hybridoma cell line with preservation number CGMCC No.46223. Ascites fluid is collected after injection, purified, and the obtained monoclonal antibody is stored at low temperature.

[0039] This invention provides the application of the method for preparing the methylphenidate monoclonal antibody in the recognition of methylphenidate.

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

[0041] (1) The methylphenidate monoclonal antibody obtained in this invention has good detection sensitivity (IC50) for methylphenidate. 50 The value was 1.02 ng / mL.

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

[0043] Preservation of biological materials

[0044] A hybridoma cell line, MOD, secreting methylphenidate monoclonal antibody, has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. It has been classified as a monoclonal cell line, deposited on October 31, 2024, with accession number CGMCC No. 46223. Attached Figure Description

[0045] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0046] Figure 1 shows the standard curve of inhibition of methylphenidate by the monoclonal antibody of methylphenidate of the present invention. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0048] The culture media involved in the following examples are as follows:

[0049] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cysteine ​​dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5, L-tyrosine 23. 19. L-Valine 20. Para-aminobenzoic acid 1. Calcium nitrate 100. Anhydrous magnesium sulfate 48.84. Anhydrous sodium dihydrogen phosphate 676.13. Potassium chloride 400. Sodium chloride 6000. Glucose 2000. Reduced glutathione 1. Phenol red 5. L-glutamine 300. Biotin 0.2. D-calcium pantothenate 0.25. Folic acid 1. I-inositol 35. Nicotinamide 1. Choline chloride 3. Pyridoxine hydrochloride 1. Riboflavin 0.2. Thiamine hydrochloride 1. Vitamin B12 0.005. Sodium bicarbonate 2000.

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

[0051] Carbonate buffer (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800 mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000 mL. Store at 4°C for later use.

[0052] Phosphate-buffered saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, 2.9g Na2HPO4·12H2O, dissolved in 800mL pure water, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000mL.

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

[0054] Antibody dilution buffer: PBS containing 0.1% gelatin;

[0055] TMB colorimetric solution: Solution A: Na₂HPO₄ 4. 12H₂O 18.43g, citric acid 9.33g, diluted to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution, and mix again before use.

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

[0057] Method for detecting methylphenidate inhibition rate: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. The antigen was diluted to 0.01 μg / mL, 0.03 μg / mL, 0.1 μg / mL, and 0.3 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.03 μg / mL, 0.1 μg / mL, 0.3 μg / mL, and 1 μg / mL with antibody dilution buffer. After selecting the optimal operating point, methylphenidate standard was diluted to eight concentrations (0, 0.3 ng / mL, 1 ng / mL, 3 ng / mL, 9 ng / mL, 27 ng / mL, and 81 ng / mL). Following the ic-ELISA procedure, an ic-ELISA curve was plotted using OriginPro 8.5 (results shown in Figure 1) to obtain the methylphenidate standard inhibition curve, and the IC50 was calculated. 50 .

[0058] Example 1: Synthesis of methylphenidate hapten

[0059] Since methylphenidate is a small molecule and lacks immunogenicity, it cannot stimulate an immune response in mice to produce antibodies. Therefore, it is necessary to use protein conjugation technology to couple methylphenidate to proteins to acquire immunogenicity. Commonly used active groups in protein conjugation technology include amino, carboxyl, hydroxyl, and thiol groups. However, since methylphenidate does not contain these active groups in its molecular structure, methylphenidate is derivatized.

[0060] 5-(tert-butylcarbonyl)aminovaleric acid (4 g, 0.0183 mol) was added to a THF solution (54 mL), followed by CDI (3.26 g, 0.02013 mol). The mixture was stirred at 15 °C for 1 hour (solution a). Methyl 2-(4-bromophenyl)acetate (12.58 g, 0.0549 mol) was dissolved in THF (90 mL), and LDA (2 M, 27.4 mL, 0.0549 mol) was added dropwise at -40 °C. After addition, the mixture was stirred at the same temperature for 1.5 hours, and the reaction was quenched with a saturated ammonium chloride aqueous solution. The pH was adjusted to 4-5 by adding 1.0 M HCl. The mixture was extracted with ethyl acetate, and the bound organic layer was washed with brine, dried over anhydrous Na₂SO₄, and concentrated under vacuum. The residue was purified by silica gel flash column chromatography to give intermediate 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). The pH was adjusted to 10 with sodium sulfate (Sat. Na₂CO₃) and then separated. 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%), a yellow solid.

[0062] Intermediate 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 a N2 atmosphere for 16 h. The mixture was diluted with H2O (100 mL), extracted with EA (100 mL), washed with brine (60 mL), dried, and concentrated. The residue was purified by silica gel flash column evaporation (EA in PE was 15%) to give intermediate 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 intermediate 3 (350 mg, 0.8383 mmol). The reaction mixture was stirred at 60 °C under a H₂ atmosphere for 16 hours. The reaction mixture was filtered to remove the catalyst, and the filtrate was concentrated and dried to obtain crude oil. The residue was purified by Prep-HPLC (NH₄HCO₃ as an additive) to give the final product as a white solid (70 mg, yield: 25%), namely methylphenidate hapten.

[0064] Example 2: Synthesis of methylphenidate complete antigen

[0065] Weigh 6 mg of methylphenidate hapten and 4.4 mg of N-hydroxysuccinimide (NHS), dissolve them in 200 μL of N,N-dimethylformamide (DMF), and stir at room temperature for 10 min. Then weigh 7.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and add it to the methylphenidate hapten solution, stirring at room temperature for 6-8 h to activate it. Take 6 mg of keyhole hemocyanin (KLH), add it to 3 mL of 0.01 M carbonate buffer (CBS), dissolve it completely, and slowly add the activated hapten to the diluted KLH solution, stirring overnight at room temperature. Then dialyze with 0.01 M PBS to remove unreacted small molecules, obtaining a relatively pure complete antigen, which is then identified by UV absorption scanning.

[0066] Example 3: Synthesis of methylphenidate-coated antigen

[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 reacted with the solution at room temperature for 10 min. 4.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in the above solution and reacted with the solution at room temperature for 6-8 h to obtain the 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 dilution solution and stirred overnight at room temperature. Then, the reaction solution was dialyzed against 0.01 M PBS to remove unreacted small molecules, yielding the coating antigen.

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

[0069] 1. Acquisition of immunity in animals

[0070] Methylphenidate complete antigen was emulsified with an equal volume of Freund's adjuvant and then administered to BALB / c mice via subcutaneous injection at multiple sites on the neck and back (except for sprint immunization). The initial immunization used complete Freund's adjuvant at a dose of 100 μg / mouse. Multiple booster immunizations used incomplete Freund's adjuvant at half the dose (50 μg / mouse). Sprint immunizations did not use adjuvant; the adjuvant was diluted directly with physiological saline and injected intraperitoneally at a dose halved again (25 μg / mouse). The interval between the initial and second booster immunizations was one month, the interval between multiple booster immunizations was 21 days, and the interval between the sprint immunization and the final booster immunization was 18-21 days. The immunization effect in mice was observed using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), which detected the serum titer and inhibition.

[0071] 2. Cell fusion

[0072] Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 4000) method, with the following specific steps:

[0073] a. After euthanizing the mouse by tail dislocation and cervical dislocation, immediately disinfect the mouse in 75% alcohol for about 5 minutes. Under aseptic conditions, remove the spleen and grind it moderately with the rubber tip of a syringe and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect the suspension, centrifuge (1200 rpm, 8 minutes), 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 them aside for later use.

[0074] b. Collection of SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells are expanded in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The number of SP2 / 0 tumor cells should reach 1-4 × 10⁶ cells before fusion. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion, tumor cells are collected and suspended in RPMI-1640 basal culture medium for cell counting during fusion.

[0075] c. Fusion process (7 min): At min 1, add 1 mL of PEG 4000 to the cells dropwise, gradually increasing the speed; at min 2, allow to stand; at min 3 and min 4, add 1 mL of RPMI-1640 medium dropwise over 1 min; at min 5 and min 6, add 2 mL of RPMI-1640 medium dropwise over 1 min; at min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s. Except for min 2, continuously agitate the solution. Then incubate at 37°C for 5 min; centrifuge (800 rpm, 8 min), discard the supernatant, and resuspend in RPMI-1640 selection medium containing 20% ​​fetal bovine serum and 2% 50×HAT. Add 200 μL / well to a 96-well cell plate and incubate at 37°C in a 5% CO2 incubator.

[0076] 3. Cell selection and cell line establishment

[0077] On day 3 after cell fusion, the fused cells were partially replaced with RPMI-1640 selection medium. On day 5, the medium was completely replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On day 7, the cell supernatant was collected for screening.

[0078] The screening process consists of two steps: First, positive cell wells are selected using ic-ELISA. Second, methylphenidate is used as a standard, and the inhibitory effect on positive cells is determined using ic-ELISA.

[0079] Cell wells that showed good inhibition of methylphenidate standard were selected, and subcloning was performed using the limiting dilution method. The cells were then tested using the same method after seven days.

[0080] At least three subcloning operations were performed using the method described above to finally obtain the methylphenidate monoclonal antibody cell line.

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

[0082] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6 Methylphenidate hybridoma cells were used, and ascites fluid was collected starting from day 7. The ascites fluid was then purified for antibody using the caprylic acid-saturated ammonium sulfate method.

[0083] 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℃.

[0084] The IC50 of methylphenidate monoclonal antibody was measured using an indirect competitive ELISA. 50 The value is 1.02 ng / mL, indicating good sensitivity to methylphenidate, and it can be used for the immunoassay detection of methylphenidate.

[0085] Example 6: Application of methylphenidate monoclonal antibody

[0086] The monoclonal antibody prepared from hybridoma cell lines via in vivo ascites fluid was used in an ELISA addition and recovery assay for methylphenidate. The specific steps are as follows:

[0087] (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.

[0088] (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;

[0089] (3) Prepare methylphenidate standard solutions of 0, 0.3 ng / mL, 1 ng / mL, 3 ng / mL, 9 ng / mL, 27 ng / mL and 81 ng / mL with phosphate buffer (PBS). Add the standard solutions and the extracts of the samples to be tested to the sealed microplates, 50 μL per well, and repeat each sample in 3 wells. Then add 50 μL of methylphenidate monoclonal antibody diluted to 0.1 μg / mL to each well. After reacting 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 with PBS containing 0.1% gelatin to each well, react at 37°C for 30 min, then wash the plate and pat dry.

[0091] (5) Add 100 μL of TMB colorimetric solution to each well, develop the color at 37℃ 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) Add 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 sample, respectively (based on the antibody linear range and IC50). 50 Set the desired concentration, and mix thoroughly using an electric shaker. Recovery experiments were conducted using an indirect competitive ELISA, with recoveries of 106%, 93%, and 114%, respectively.

[0095] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A hybridoma cell line secreting methylphenidate monoclonal antibody, characterized in that, The hybridoma cell line was deposited on October 31, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46223, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as a monoclonal cell line. The structural formula of the methylphenidate hapten used to prepare the hybridoma cell line is as follows: 。 2. A methylphenidate monoclonal antibody, characterized in that, Produced by the hybridoma cell line described in claim 1.

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

4. A kit for detecting methylphenidate, characterized in that, The kit contains the methylphenidate monoclonal antibody as described in claim 2.

5. The reagent kit according to claim 4, characterized in that, The kit is selected from enzyme-linked immunosorbent assay (ELISA) kits, fluorescence immunoassay kits, or chemiluminescence immunoassay kits.

6. A test strip for detecting methylphenidate, characterized in that, The test strip contains the methylphenidate monoclonal antibody as described in claim 2.

7. The test strip according to claim 6, characterized in that, The test strip includes a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane has a detection line and a control line arranged sequentially on it. The conjugate pad is coated with the methylphenidate monoclonal antibody as described in claim 2.

8. A chip for detecting methylphenidate, characterized in that, The chip contains the methylphenidate monoclonal antibody as described in claim 2.

9. The use of the hybridoma cell line of claim 1, the methylphenidate monoclonal antibody of claim 2, the composition of claim 3, the kit of any one of claims 4-5, the test strip of any one of claims 6-7, or the chip of claim 8 in the detection of methylphenidate.

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