Hybridoma cell strain secreting monoclone antibody of clozapine and its metabolite

Abstract

The present application relates to a hybridoma cell strain secreting clozapine and its metabolite monoclonal antibody, and belongs to the technical field of immune detection. The present application prepares a hapten by chemically synthesizing clozapine and its metabolite, and then prepares a complete antigen, and then screens a hybridoma cell strain secreting clozapine and its metabolite monoclonal antibody through animal immunization and cell fusion. The hybridoma cell strain LDP-2B10 provided by the present application has been preserved in the China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC No. 46233. The monoclonal antibody secreted by the hybridoma cell strain has good sensitivity and specificity, the IC 50 value of the monoclonal antibody for clozapine is 5.041 ng / mL, the IC 50 value of the monoclonal antibody for the metabolite N-desmethylclozapine is 5.524 ng / mL, and the IC 50 value of the monoclonal antibody for the metabolite clozapine-N-oxide is 5.551 ng / mL. The cross reactivity of the monoclonal antibody for various clozapine functional analogues is less than 1%, and the monoclonal antibody can be used for detecting clozapine in urine and blood drug concentration, and has practical application value.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a hybridoma cell line that secretes monoclonal antibodies against clozapine and its metabolites. Background Technology

[0002] Clozapine, also known as 8-chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo[b,e][1,4]diazapine Clozapine, with the molecular formula C18H19ClN4, a molecular weight of 326.823, and CAS number 5786-21-0, is an antipsychotic drug primarily used to treat treatment-resistant schizophrenia. Studies have found that clozapine can effectively control positive symptoms such as hallucinations and delusions and prevent suicidal behavior, leading to its widespread clinical application. In clinical use, the dosage of clozapine is closely correlated with the patient's blood drug concentration. Different patients have varying metabolic capacities for clozapine due to individual differences such as age, weight, liver and kidney function, and genetic polymorphisms. The same dosage may produce significantly different blood drug concentrations in different patients. If the blood drug concentration is too low, the drug cannot exert its full therapeutic effect, resulting in ineffective disease control; while if the blood drug concentration is too high, it may cause various adverse reactions, such as drowsiness, constipation, and granulocytopenia, and in severe cases, may even endanger the patient's life.

[0003] To further explore the clinical significance of clozapine drug concentration monitoring and to comprehensively understand its metabolism in the body, it is crucial to measure the concentrations of both the parent drug and its active metabolites. This not only helps to accurately grasp the drug's mechanism of action in patients but also provides solid data support for rational drug use in clinical practice, thereby further improving treatment efficacy and safety.

[0004] Currently, there are limited studies on analytical methods for determining clozapine content, mainly relying on traditional methods such as high-performance liquid chromatography (HPLC-UV), liquid chromatography-tandem mass spectrometry (LC-MS / MS), and chemical titration. These methods suffer from drawbacks such as being time-consuming, costly, cumbersome in operation, and unsuitable for rapid on-site detection. Therefore, establishing a rapid and convenient method for detecting clozapine and its metabolites is of great significance. Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method suitable for rapid on-site detection of large numbers of samples. It can specifically identify target analytes in complex biological sample mechanisms (such as blood and urine), providing a new detection route for clozapine and its metabolites. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes monoclonal antibodies against clozapine and its metabolites. This invention involves chemically synthesizing clozapine and its metabolite haptens to prepare complete antigens, followed by animal immunization and cell fusion to screen and obtain a hybridoma cell line that secretes monoclonal antibodies against clozapine and its metabolites. The hybridoma cell line LDP-2B10 provided by this invention has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46233.

[0006] The first objective of this invention is to provide a hybridoma cell line that secretes monoclonal antibodies against clozapine and its metabolites, the hybridoma cell line having the accession number CGMCC NO.46233.

[0007] Furthermore, the method for preparing the hybridoma cell line includes the following steps:

[0008] Step S1: Prepare clozapine and its metabolite hapten and clozapine and its metabolite complete antigen. Prepare the obtained clozapine and its metabolite complete antigen into Freund's complete adjuvant containing antigen and Freund's incomplete adjuvant containing antigen.

[0009] Step S2: Perform initial immunization, booster immunization, and sprint immunization on the immunized animals. The initial immunization uses Freund's complete adjuvant containing antigen as described in S1. The booster immunization uses Freund's incomplete adjuvant containing antigen as described in step S1. The sprint immunization uses clozapine and its metabolites as complete antigen as described in S1.

[0010] Step S3: Take spleen cells and myeloma cells from the immunized animals after sprint immunization in step S2 and perform cell fusion. Culture the fused cells in HAT medium and detect positive cell pores using indirect ELISA. Further determine the inhibitory effect of positive cell pores using indirect competitive ELISA. Subclone the positive cell pores with the best inhibition using limiting dilution method to obtain hybridoma cell lines.

[0011] Furthermore, the structural formulas of the clozapine and its metabolite hapten are shown below:

[0012]

[0013] Further, in step S1, the method for preparing the clozapine and its metabolite hapten includes the following steps:

[0014] 425 mg of clozapine was weighed and dissolved in 0.4 mL of N,N-dimethylformamide. 538 mg of ethyl 3-hydroxypropionate and 51.2 mg of potassium carbonate were added and the mixture was stirred at 80 °C for 12 hours. After the reaction was completed, the mixture was cooled to room temperature and extracted three times with 20 mL of dichloromethane. The organic phases were combined, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain clozapine and its metabolite hapten.

[0015] Further, in step S1, the method for preparing the complete antigen of clozapine and its metabolites includes the following steps:

[0016] The clozapine and its metabolite hapten, sodium nitrite and hydrochloric acid are mixed and dissolved in a solvent, and the mixture is reacted to obtain a mixed solution. The mixed solution is then added to a carrier protein solution to react and obtain the complete antigen of clozapine and its metabolite.

[0017] Furthermore, in step S2, the initial immunization and booster immunization are administered to the immunized animal via subcutaneous injection on the back; the sprint immunization is administered to the immunized animal via intraperitoneal injection.

[0018] Furthermore, in step S2, during the enhanced immunization process, blood is collected from the immunized animals, and the serum titer and inhibition rate are detected by indirect competitive enzyme-linked immunosorbent assay (ELISA) to screen out immunized animals with high titer and good inhibition.

[0019] Furthermore, in step S3, the cell fusion is performed using the polyethylene glycol (PEG 1500) method.

[0020] A second objective of this invention is to provide the application of the above-mentioned hybridoma cell line in the preparation of monoclonal antibodies against clozapine and its metabolites.

[0021] Furthermore, it includes the following steps:

[0022] Sterile paraffin oil was injected into the peritoneum of immunized animals, followed by the injection of the hybridoma cell line provided by this invention (CGMCC No. 46233). Ascites fluid was collected after injection, purified, and monoclonal antibodies against clozapine and its metabolites were obtained and stored at low temperature.

[0023] A third objective of this invention is to provide a monoclonal antibody against clozapine and its metabolites, wherein the monoclonal antibody against clozapine and its metabolites is secreted by the aforementioned hybridoma cell line.

[0024] Furthermore, the metabolite is N-demethylclozapine and / or clozapine-N-oxide.

[0025] A fourth object of the present invention is to provide a composition comprising the above-described hybridoma cell line and / or the above-described monoclonal antibody.

[0026] A fifth objective of this invention is to provide the use of the above-mentioned hybridoma cell line, the above-mentioned monoclonal antibody against clozapine and its metabolites, or the above-mentioned composition in the preparation of detection products for clozapine and its metabolites.

[0027] A sixth object of the present invention is to provide a test strip containing the above-mentioned hybridoma cell line, the above-mentioned clozapine and its metabolite monoclonal antibody, or the above-mentioned composition.

[0028] Furthermore, 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 monoclonal antibodies against clozapine and its metabolites, and the monoclonal antibodies against clozapine and its metabolites are labeled with colloidal gold or fluorescent microspheres.

[0029] A seventh object of the present invention is to provide a kit containing the above-described hybridoma cell line, the above-described clozapine and its metabolite monoclonal antibody, or the above-described composition.

[0030] Furthermore, the kit also includes an enzyme-labeled plate, clozapine and its metabolites coated antigen, clozapine and its metabolites standard solution, enzyme-labeled secondary antibody, and colorimetric solution.

[0031] An eighth object of the present invention is to provide the use of the above-described hybridoma cell line, the above-described monoclonal antibody, the above-described composition, the above-described test strip, or the above-described kit in the detection of clozapine and its metabolites.

[0032] The beneficial effects of this invention are:

[0033] The monoclonal antibody secreted by the tumor cell line provided by this invention exhibits good broad-spectrum specificity (cross-cross rates of 94.7% and 94.2% for clozapine metabolites N-demethylclozapine and clozapine-N-oxide, respectively, and less than 1% for other psychotropic drugs olanzapine, risperidone, and ziprasidone) and detection sensitivity (IC50). 50 The values ​​were 5.041 ng / mL, which can be used to detect the residues of clozapine and its metabolites in brown rice and soybeans. This provides raw materials for the immunological detection of clozapine and its metabolites in food and has practical application value.

[0034] Preservation of biological materials

[0035] A hybridoma cell line, LDP-2B10, which secretes monoclonal antibodies against clozapine and its metabolites, has been deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. It is classified as a monoclonal cell line, deposited on October 31, 2024, with accession number CGMCC No. 46233. Attached Figure Description

[0036] 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...

[0037] Figure 1 The standard curve of clozapine and its metabolite monoclonal antibody inhibition against clozapine provided by the present invention;

[0038] Figure 2 The standard curve of inhibition of N-demethylclozapine by monoclonal antibodies against clozapine and its metabolites provided by the present invention;

[0039] Figure 3 The standard curve of inhibition of clozapine and its metabolite monoclonal antibody against clozapine-N-oxide provided by the present invention. Detailed Implementation

[0040] 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.

[0041] The culture media involved in the following examples are as follows:

[0042] 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.

[0043] The reagents involved in the following examples are as follows:

[0044] Carbonate buffer (CBS): Weigh 1.59g of Na2CO3 and 2.93g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800mL and mix well. Adjust the pH to 9.6 and add double-distilled water to make up to 1000mL. Store at 4℃ for later use.

[0045] 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 with ultrapure water.

[0046] Washing buffer (PBST): Add 0.5 mL of Tween-20 to 1000 mL of 0.01 mol / L pH 7.4 PBS solution;

[0047] PBST: PBS containing 0.05% Tween-20;

[0048] Antibody diluent: a washing solution containing 0.1% gelatin;

[0049] TMB colorimetric solution: Solution A: 18.43g Na2HPO4·12H2O, 9.33g citric acid, diluted to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solutions A and B in a volume ratio of 5:1 to obtain the TMB colorimetric solution. Mix fresh before use.

[0050] The detection methods involved in the following embodiments are as follows:

[0051] Clozapine inhibition rate assay: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. The antigen was diluted to 1, 0.3, 0.1, and 0.03 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 1, 0.3, 0.1, and 0.03 μg / mL with antibody dilution buffer. After selecting the optimal operating point, clozapine standards were diluted to concentrations of 100, 33.33, 11.11, 3.7, 1.23, 0.41, 0.13, and 0 ng / mL. Following the ic-ELISA procedure, the clozapine standard inhibition curve was plotted using Origin 2024, and the IC50 was calculated. 50 .

[0052] Example 1: Preparation of complete antigen

[0053] 1. The synthesis of a hapten is shown in the following reaction equation:

[0054]

[0055] The derivation steps are briefly described below:

[0056] Preparation of clozapine and its metabolite hapten: 425 mg of clozapine was dissolved in 0.4 mL of N,N-dimethylformamide, 538 mg of ethyl 3-hydroxypropionate and 512 mg of potassium carbonate were added, and the mixture was stirred at 80 °C for 12 hours. After the reaction was completed, the mixture was cooled to room temperature, extracted three times with 20 mL of dichloromethane, and the organic phases were combined, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain clozapine and its metabolite hapten LDP-COOH.

[0057] 2. Weigh 9 mg of the prepared LDP-COOH and dissolve it in 200 μL of DMF. Add 5.89 mg of N-hydroxysuccinimide (NHS) and react for 30 minutes with stirring at 25 °C. Then add 11.12 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and react for 6 hours. The resulting mixture is called solution A. Then weigh 10 mg of keyhole hemocyanin (KLH) and dissolve it in 2 mL of carbonate buffer. This is called solution B. Slowly add solution A to solution B and react at room temperature with stirring for 12 hours. Dialyze the mixture with 0.01 mol / L phosphate-buffered saline (PBS) for 3 days to obtain the conjugate clozapine-KLH. Store at -20 °C for later use.

[0058] Example 2: Preparation of the coating agent

[0059] Weigh 8 mg of the prepared LDP-COOH and dissolve it in 200 μL of DMF. Add 4.78 mg of N-hydroxysuccinimide (NHS) and react for 30 minutes with stirring at 25 °C. Then add 9.32 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and react for 6 hours. The resulting mixture is called solution A. Then weigh 10 mg of chicken ovalbumin (OVA) and dissolve it in 2 mL of carbonate buffer. This is called solution B. Slowly add solution A to solution B and react at room temperature with stirring for 12 hours. Dialyze the mixture with 0.01 mol / L phosphate-buffered saline (PBS) for 3 days to obtain the conjugate clozapine-OVA. Store at -20 °C for later use.

[0060] Example 3: Preparation of hybridoma cell lines

[0061] I. Mouse Immunization

[0062] Healthy BALB / c mice aged 6-8 weeks were selected for immunization. Clozapine and its metabolite complete antigen were emulsified with an equal volume of Freund's adjuvant and administered subcutaneously via dorsal injection to BALB / c mice. The first immunization used complete Freund's adjuvant, and subsequent immunizations used incomplete Freund's adjuvant. There was a 28-day interval between the first and second booster immunizations, and a 21-day interval between multiple booster immunizations. Seven days after the third immunization, blood was collected (5 μL of blood from tail amputation + 995 μL of antibody diluent = antiserum). Serum titers and inhibition were measured using ic-ELISA. Mice with high titers and good inhibition were selected for a fifth immunization 21 days after the fifth immunization, administered via intraperitoneal injection at half the dose and without any adjuvant.

[0063] II. Cell Fusion

[0064] Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 1500) method, with the following specific steps:

[0065] (1) After euthanizing the mice by enucleation and cervical dislocation, the mice were immediately disinfected in 75% alcohol and soaked for about 5 minutes. The spleen of the mice was removed under aseptic conditions. The spleen cells were moderately ground with the rubber tip of a syringe and passed through a 200-mesh cell sieve to obtain a suspension of spleen cells. The suspension was collected and centrifuged (1200 rpm, 8 min). The spleen cells were washed three times with RPMI-1640 medium. After the last centrifugation, the spleen cells were diluted to a certain volume, counted, and set aside for later use.

[0066] (2) Collection of murine myeloma SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The required number of SP2 / 0 tumor cells before fusion was (1-4) × 10⁻⁶. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. During fusion, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counting is performed.

[0067] (3) Fusion process: 7 min. At min 1, 1 mL of PEG 1500 was added to the cells dropwise, gradually increasing the speed. At min 2, the cells were allowed to stand. At min 3 and min 4, 1 mL of RPMI-1640 medium was added dropwise over 1 min. At min 5 and min 6, 2 mL of RPMI-1640 medium was added dropwise over 1 min. At min 7, 1 mL of RPMI-1640 medium was added dropwise every 10 s. The cells were then incubated at 37°C for 5 min. After centrifugation (800 rpm, 8 min), the supernatant was discarded, and the cells were resuspended in RPMI-1640 selection medium containing 20% ​​fetal bovine serum and 2% 50×HAT. The medium was added to 96-well cell plates at a rate of 200 μL / well and incubated at 37°C in a 5% CO2 incubator.

[0068] III. Cell Screening and Cell Line Establishment

[0069] On day 3 of cell fusion, the fused cells underwent a half-medium replacement 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 selection. The selection process consisted of two steps: first, positive cell wells were selected using ic-ELISA; second, clozapine was used as a standard, and its inhibitory effect on positive cells was determined using ic-ELISA. Cell wells showing good inhibition against clozapine standard were selected, and subcloning was performed using the limiting dilution method. The same method was repeated three times to obtain cell lines.

[0070] Example 4: Preparation and Identification of Monoclonal Antibodies

[0071] 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 Hybridoma cells were used, and ascites fluid was collected starting on day 7. The ascites fluid was purified using the caprylic acid-ammonium sulfate method. Under slightly acidic conditions, caprylic acid precipitates other proteins in the ascites fluid besides IgG immunoglobulins. The precipitate was then discarded by centrifugation. IgG-type monoclonal antibodies were then precipitated with an equal volume of saturated ammonium sulfate solution, centrifuged, and the supernatant was discarded. The precipitate was dissolved in 0.01M PBS solution (pH=7.4), dialyzed to desalt, and finally the purified monoclonal antibodies were stored at -20°C.

[0072] The IC50 of the monoclonal antibody against clozapine was determined using an indirect competitive ELISA method. 50 The concentration was 5.041 ng / mL, and its IC50 for metabolites and analogues was verified. 50 The cross-reactivity rates were 94.7% and 94.2% with clozapine's metabolites N-demethylclozapine and clozapine-N-oxide, respectively, and less than 1% with other psychotropic drugs olanzapine, risperidone, and ziprasidone. The cross-reactivity rate was calculated as follows: (IC50 of clozapine) / (C100%). 50 / metabolites or similar IC 50 The cross-reactivity ratio (CRR) is calculated as 100%. Based on the CRR values, it can be seen that the antibody has high sensitivity and specificity to clozapine and its metabolites, as shown in Table 1.

[0073] Table 1. IC50 of monoclonal antibodies against clozapine, its metabolites, and analogues. 50 and cross-reactivity rate

[0074] name <![CDATA[IC 50 (ng / mL)]]> Cross-reactivity rate (%) Clozapine 5.041 100 N-Desmethylclozapine 5.524 94.7 Clozapine-N-oxide 5.551 94.2 Olanzapine >100 <1 Risperidone >100 <1 Ziprasidone >100 <1

[0075] Example 5: Application of Monoclonal Antibodies

[0076] Monoclonal antibodies prepared from hybridoma cell lines via in vivo ascites fluid were used in a clozapine spiking and recovery assay. The specific steps are as follows:

[0077] 1. Coating: The coated clozapine-OVA was serially diluted with 0.05M pH=9.6 carbonate buffer, starting from 1 μg / mL, 100 μL / well, and reacted at 37℃ for 2 h.

[0078] 2. Washing: Pour off the solution in the plate and wash with washing solution 3 times, 3 minutes each time.

[0079] 3. Sealing: After patting dry, add 200 μL / well sealing solution and react at 37℃ for 2 h. Wash and dry for later use.

[0080] 4. Sample addition: Serially dilute the antiserum (antiserum obtained by diluting mouse tail blood with antibody diluent) starting from 1:1000 and add it to each well of the coated sample at 100 μL / well. Incubate at 37°C for 30 min. After thorough washing, add 1:3000 diluted HRP-goat anti-mouse IgG at 100 μL / well and incubate at 37°C for 30 min.

[0081] 5. Color development: Remove the microplate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37°C in the dark for 15 min.

[0082] 6. Termination and Measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 value.

[0083] The standard curves of clozapine inhibition by monoclonal antibodies against clozapine are as follows: Figure 1 As shown, the IC50 of the monoclonal antibody against clozapine was determined by ic-ELISA. 50The concentration was 5.041 ng / mL, indicating good sensitivity to clozapine. For recovery, urine and plasma samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). LC-MS / MS analysis showed no clozapine residues in any of the three samples; therefore, this study determined the recovery rate by adding clozapine and its metabolites. Specifically, as shown in Table 2, the average recovery rate for clozapine-based LC-MS / MS was 97.8-116.8%, with a relative standard deviation (RSD) of 2.32-7.87%; the recovery rate for the indirect competitive ELISA method was 94.7-113.3%, with an RSD of 2.78-6.33%. The average recovery rate for N-demethylclozapine-based LC-MS / MS was 93.2-109.9%, with a relative standard deviation (RSD) of 2.17-6.54%; the recovery rate for the indirect competitive ELISA method was... The recoveries of clozapine-N-oxide-based LC-MS / MS were 90.1-117.3%, with RSDs of 3.12-6.56%. The recoveries of clozapine-N-oxide-based LC-MS / MS were 94.4-114.2%, with RSDs of 2.27-6.28%. This indicates that the method for detecting clozapine based on monoclonal antibodies against clozapine and its metabolites is relatively accurate and can be used for immunoassay detection of clozapine residues in plasma and urine.

[0084] Table 2 Recovery rates of monoclonal antibodies against clozapine and its metabolites in urine and plasma

[0085]

[0086]

[0087] 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 that secretes monoclonal antibodies against clozapine and its metabolites, characterized in that: The hybridoma cell line has the accession number CGMCC NO.46233.

2. The use of the hybridoma cell line of claim 1 in the preparation of monoclonal antibodies against clozapine and its metabolites.

3. A monoclonal antibody against clozapine and its metabolites, characterized in that: The clozapine and its metabolite monoclonal antibody are obtained from the hybridoma cell line described in claim 1.

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

5. The use of the hybridoma cell line of claim 1, the monoclonal antibody against clozapine and its metabolites of claim 3, or the composition of claim 4 in the preparation of a detection product for clozapine and its metabolites, characterized in that, The metabolites are N-demethylclozapine and / or clozapine-N-oxide.

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

7. A reagent kit, characterized in that: The kit contains the hybridoma cell line of claim 1, the monoclonal antibody against clozapine and its metabolites of claim 3, or the composition of claim 4.

8. The reagent kit according to claim 7, characterized in that: The kit also includes an enzyme-labeled plate, clozapine and its metabolites coated antigen, clozapine and its metabolites standard solution, enzyme-labeled secondary antibody, and colorimetric solution.

9. The use of the hybridoma cell line of claim 1, the monoclonal antibody of claim 3, the composition of claim 4, the test strip of claim 6, or the kit of claim 7 or 8 in the detection of clozapine and its metabolites, characterized in that, The metabolites are N-demethylclozapine and / or clozapine-N-oxide.

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