Hybridoma cell strain secreting olanzapine compound monoclonal antibody

Abstract

The invention relates to a hybridoma cell strain secreting an olanzapine compound monoclonal antibody. The hybridoma cell strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on October 31, 2024, the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.46224. According to the present invention, the monoclonal antibody secreted by the hybridoma cell strain has good sensitivity on the olanzapine compound, the IC50 value of the olanzapine is 4.18 ng / mL, the IC50 value of the clozapine is 6.69 ng / mL, and the IC50 value of the quetiapine is 9.98 ng / mL, such that the olanzapine compound can be efficiently and rapidly detected.

Description

Technical Field

[0001] The present invention relates to the technical field of immunochemistry, and in particular to a hybridoma cell line secreting monoclonal antibodies against olanzapine compounds. Background Art

[0002] Olanzapine compounds mainly include olanzapine, quetiapine and clozapine, all of which are antipsychotic drugs. Among them, the English generic name of olanzapine is Olanzapine, and its chemical name is 2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-b][1,5]benzodiazepine. It has affinity for multiple receptors such as 5-HT, dopamine D, α-adrenaline, histamine H, etc., and its affinity for 5-HT2 receptors in vitro and in vivo is greater than its affinity for dopamine D2 receptors; the English generic name of quetiapine is Quetiapine, and its chemical name is 11-[4-[2-(2-hydroxyethoxy)ethyl]-1-piperazinyl]dibenzo[b,f][1,4]thiazepine; the English generic name of clozapine is Clozapine, and its chemical name is 8-chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine In clinical practice, olanzapine compounds are often used to control agitation symptoms in patients with schizophrenia, bipolar mania and dementia. They can significantly improve the negative (e.g., apathy, emotional and social withdrawal, poverty of speech) and positive symptoms (e.g., delusions, hallucinations, thought disorders, hostility and suspicion) of schizophrenia, and can also relieve common secondary affective symptoms of schizophrenia and related diseases.

[0003] At present, the detection method of olanzapine compounds is mainly instrumental detection, and the commonly used methods are gas chromatography, liquid chromatography and gas chromatography-mass spectrometry. Although these chromatography-based methods have high sensitivity and specificity, they have some 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 olanzapine residues is needed.

[0004] Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method. It has simple sample pretreatment, few purification steps, large analysis capacity, low detection cost and easy operation. 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 ELISA to detect olanzapine compounds is to obtain monoclonal antibodies with high sensitivity to olanzapine compounds, but the existing technology lacks monoclonal antibodies with high sensitivity to olanzapine compounds. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art of lacking a monoclonal antibody with high sensitivity to olanzapine compounds.

[0006] In order to solve the above technical problems, the present invention provides a hybridoma cell line that secretes monoclonal antibodies against olanzapine compounds. The monoclonal antibodies secreted by the hybridoma cell line of the present invention have good sensitivity to olanzapine compounds, such as olanzapine, clozapine, and quetiapine, among which the IC of olanzapine is 50 The value is 4.18ng / mL, and the IC value of clozapine 50 The value was 6.69ng / mL, and the IC value of quetiapine 50 The value is 9.98ng / mL, so low concentrations of olanzapine compounds can be detected simultaneously.

[0007] The first object of the present invention is to provide a hybridoma cell strain, which was deposited in the General Microbiology Center of China Culture Collection Administration on October 31, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 46224.

[0008] Furthermore, the hybridoma cell line is obtained by immunizing an animal with a complete antigen prepared by a hapten, wherein the structural formula of the hapten is as shown in Formula I:

[0009]

[0010] Furthermore, the complete antigen is obtained by coupling the hapten to a carrier protein.

[0011] Furthermore, the carrier protein includes keyhole limpet hemocyanin.

[0012] The second object of the present invention is to provide a use of the hybridoma cell line in detecting olanzapine compounds.

[0013] Furthermore, the olanzapine compounds include olanzapine, clozapine and quetiapine.

[0014] The third object of the present invention is to provide a monoclonal antibody secreted by the hybridoma cell line.

[0015] The fourth object of the present invention is to provide a use of the above monoclonal antibody in detecting olanzapine compounds.

[0016] A fifth object of the present invention is to provide a detection product for olanzapine compounds, wherein the detection product comprises the above-mentioned monoclonal antibody.

[0017] Furthermore, the detection product also includes a coating source.

[0018] Furthermore, the coating agent is prepared by coupling a hapten to a carrier protein, wherein the carrier protein comprises bovine serum albumin.

[0019] Beneficial effects of the present invention:

[0020] The monoclonal antibody secreted by the hybridoma cell line of the present invention has a high sensitivity to olanzapine compounds, wherein the IC 50 The value is 4.18ng / mL, and the IC value of clozapine 50 The value was 6.69ng / mL, and the IC value of quetiapine 50 The value is 9.98 ng / mL, so a trace amount of olanzapine compounds in the sample can be detected. The present invention provides an immunological method for detecting the content of olanzapine compounds in a sample, which has practical application value.

[0021] Biomaterial Deposit

[0022] Monoclonal cell line MEI, the monoclonal cell line was deposited in the General Microbiology Center of China Culture Collection Administration on October 31, 2024, with the deposit number CGMCC No.46224, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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

[0024] Figure 1 This is the standard inhibition curve of the monoclonal antibody against olanzapine compounds. DETAILED DESCRIPTION

[0025] 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.

[0026] The culture medium involved in the following examples is as follows:

[0027] 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, niacinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B12 0.005, sodium bicarbonate 2000.

[0028] The reagents involved in the following examples are as follows:

[0029] Carbonate buffer (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of 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.

[0030] 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;

[0031] PBST: PBS containing 0.05% Tween 20;

[0032] Antibody diluent: PBS containing 0.1% gelatin;

[0033] TMB colorimetric solution: Solution A: 18.43g Na2HPO4·12H2O, 9.33g citric acid, dilute to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solution A and solution B in a ratio of 5:1 to obtain the TMB colorimetric solution, and mix again when using.

[0034] The detection methods involved in the following embodiments are as follows:

[0035] Method for detecting the inhibition rate of olanzapine compounds: The most suitable antigen and antibody concentrations in ic-ELISA were selected by chessboard test. The antigen was diluted to 0.03, 0.1, 0.3 and 1 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.03, 0.1, 0.3 and 1 μg / mL with antibody diluent. After selecting the optimal working point, the olanzapine compound standard was diluted to 8 concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20 and 40 ng / mL), and the ic-ELISA operation steps were followed. Finally, OriginPro8.5 was used to make a graph (the results are shown in Figure 1 As shown), obtain the standard inhibition curve of olanzapine compounds and calculate IC 50 .

[0036] Example 1: Synthesis of olanzapine compound hapten

[0037] Since the olanzapine small molecule is not immunogenic and cannot stimulate mice to produce an immune response and then produce antibodies, olanzapine 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 olanzapine does not contain these active groups, olanzapine is derivatized.

[0038] 156.2 mg of olanzapine was dissolved in 1 mL of acetone, and 122.7 mg of ethyl 6-bromohexanoate, 83 mg of anhydrous potassium carbonate, and 8.3 mg of potassium iodide were added. The mixture was refluxed in a water bath at 70° C. for 48 h. The mixture was filtered and the acetone was evaporated. The residue was dissolved in 6.7 mL of ethanol and 3.3 mL of 1 M NaOH and refluxed overnight. The solution was then acidified to pH <2 with 2.5 M HCl and extracted three times with ethyl acetate. The prepared olanzapine compound hapten is shown in Formula I:

[0040]

[0041] Example 2: Synthesis of complete antigen of olanzapine compounds

[0042] Weigh 22 mg of olanzapine hapten and 6.2 mg of N-hydroxysuccinimide (NHS), dissolve in 300 μL of N,N-dimethylformamide (DMF), and stir at room temperature for 15 min; then weigh 10.35 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), add to the olanzapine hapten solution, stir at room temperature for 6-8 h, and 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.

[0043] Example 3: Synthesis of Olanzapine Compound Coating

[0044] 13 mg olanzapine hapten and 3.71 mg N-hydroxysuccinimide (NHS) were dissolved in 300 μL anhydrous N, N-dimethylformamide (DMF) and stirred at room temperature for 10 min; 6.18 mg 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 bovine serum albumin (BSA) 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 an olanzapine compound coating source.

[0045] Example 4: Preparation of hybridoma cell lines secreting monoclonal antibodies to olanzapine compounds

[0046] (1) Acquisition of animal immunity

[0047] After the complete antigen of olanzapine compounds 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); complete Freund's adjuvant was used for the first immunization, with a dose of 100 μg / mouse; incomplete Freund's adjuvant was used for multiple booster immunizations and the dose was halved to 50 μg / mouse; no adjuvant was used for sprint immunizations, and the mice were directly diluted with normal saline and injected intraperitoneally, and the dose was halved again to 25 μg / mouse; the interval between the first immunization and the second booster immunization was one month, the interval between multiple booster immunizations was 21 days, and the interval between the sprint immunization and the last booster immunization was 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;

[0048] (2) Cell fusion

[0049] 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:

[0050] 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;

[0051] 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;

[0052] 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 a 96-well cell plate at 200μL / well, and cultured in a 37℃, 5% CO2 incubator.

[0053] (3) Cell screening and cell line establishment

[0054] 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.

[0055] 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 olanzapine as the standard and use the ic-ELISA method to measure the inhibitory effect on the positive cells.

[0056] The cell wells with better inhibition of olanzapine standard were selected, subcloned by limiting dilution method, and tested by the same method seven days later.

[0057] Subcloning was performed at least three times according to the above method to finally obtain a monoclonal antibody cell line of olanzapine compounds.

[0058] Example 5: Preparation and identification of monoclonal antibodies against olanzapine compounds

[0059] 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 Olanzapine hybridoma cells, ascites was collected from the seventh day, and the ascites was used to purify antibodies by the caprylic acid-saturated ammonium sulfate method.

[0060] 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.

[0061] The IC of monoclonal antibodies against olanzapine was measured using indirect competitive ELISA. 50 The value is 4.18ng / mL, and the IC value of clozapine 50 The value was 6.69 ng / mL, and the IC value of quetiapine 50 The value was 9.98 ng / mL, indicating good sensitivity to olanzapine compounds and can be used for immunoassay detection of olanzapine compounds.

[0062] Example 6: Application of monoclonal antibodies against olanzapine compounds

[0063] Taking olanzapine as an example, the monoclonal antibody prepared by hybridoma cell line through ascites in vivo was applied to the ELISA addition recovery test of olanzapine. The specific steps are as follows:

[0064] 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 solution, 200 μL per well each time, 3 min each time, and pat dry;

[0065] 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;

[0066] Phosphate buffered saline (PBS) was used to prepare olanzapine standard solutions of 0, 0.037, 0.111, 0.333, 1, 3, 9 and 27 ng / mL, respectively. The standard solutions and the sample extracts to be tested were added to the blocked ELISA plate, 50 μL per well, and each sample was repeated for 3 wells. Then, 50 μL of olanzapine monoclonal antibody diluted to 0.1 μg / mL was added to each well. After reacting at 37°C for 30 min, the plate was washed and patted dry.

[0067] 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 in PBS containing 0.1% gelatin was added to each well, reacted at 37°C for 30 min, and the plate was washed and patted dry;

[0068] Add 100 μL of TMB colorimetric solution to each well, develop at 37°C for 15 min, then add 50 μL of 2M H2SO4 stop solution to each well, and measure absorbance at 450 nm;

[0069] Add recovery and sample preparation:

[0070] Urine was selected as the test sample.

[0071] Add 0.5 ppb, 5 ppb, and 50 ppb of olanzapine 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 109%, 89% and 113% respectively by indirect competitive ELISA.

[0072] 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, characterized in that The hybridoma cell line was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on October 31, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.46224.

2. The hybridoma cell line according to claim 1, characterized in that The hybridoma cell line is obtained by immunizing an animal with a complete antigen prepared by a hapten, wherein the structural formula of the hapten is shown in Formula I:

3. The hybridoma cell line according to claim 2, characterized in that The complete antigen is obtained by coupling the hapten to a carrier protein.

4. The hybridoma cell line according to claim 3, characterized in that The carrier protein includes keyhole limpet hemocyanin.

5. Use of the hybridoma cell line according to any one of claims 1 to 4 in detecting olanzapine compounds.

6. A monoclonal antibody secreted by the hybridoma cell line according to any one of claims 1 to 4.

7. Use of the monoclonal antibody according to claim 6 in detecting olanzapine compounds.

8. A detection product for olanzapine compounds, characterized in that: The detection product comprises the monoclonal antibody according to claim 6.

9. The detection product according to claim 8, characterized in that: The detection product also includes a coating source.

10. The detection product according to claim 9, characterized in that: The coating is prepared by coupling hapten with carrier protein, wherein the carrier protein includes bovine serum albumin.

Images