A hybridoma cell strain secreting a monoclonal antibody of olanzapine class compounds

Abstract

The present application relates to a hybridoma cell strain secreting a monoclonal antibody of olanzapine compounds. The hybridoma cell strain of the present application was deposited with the China General Microbiological Culture Collection Center on October 31, 2024, at the address of No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number of CGMCC NO. 46224. The monoclonal antibody secreted by the hybridoma cell strain of the present application has good sensitivity to olanzapine compounds, wherein the IC 50 value of olanzapine is 4.18 ng / mL, the IC 50 value of clozapine is 6.69 ng / mL, and the IC 50 value of quetiapine is 9.98 ng / mL, so that the olanzapine compounds can be detected efficiently and rapidly.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunochemistry, and particularly relates to a hybridoma cell strain secreting a monoclonal antibody of an olanzapine compound. BACKGROUND

[0002] Olanzapine compounds mainly include olanzapine, quetiapine and clozapine, and all belong to antipsychotic drugs. Among them, the English common name of olanzapine is Olanzapine, and the chemical name is 2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-b][1,5]benzodiazepine. It has affinity to 5-HT, dopamine D, alpha-adrenergic, histamine H and other receptors, and the affinity to 5-HT2 receptor in vitro and in vivo is greater than that to dopamine D2 receptor. The English common name of quetiapine is Quetiapine, and the chemical name is 11-[4-[2-(2-hydroxyethoxy)ethyl]-1-piperazinyl]dibenzo[b,f][1,4]thiopyran; the English common name of clozapine is Clozapine, and the 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 the agitation symptoms of patients with schizophrenia, bipolar mania and dementia, and can significantly improve the negative (such as affective flattening, affective and social withdrawal, and speech poverty) and positive symptoms (such as delusion, hallucination, thought disorder, hostility and suspicion) of schizophrenia, and can also alleviate the secondary affective symptoms commonly seen in schizophrenia and related diseases.

[0003] At present, the detection method of olanzapine compounds is mainly instrument 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, 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 olanzapine residues is needed.

[0004] Enzyme-linked immunosorbent assay (ELISA) is a very efficient, sensitive and rapid detection method. The sample pretreatment is simple, the purification steps are few, the analysis capacity is large, the detection cost is low and the operation is simple, which is suitable for on-site rapid detection of a large number of samples, and has been widely used in drug residue analysis. However, the premise of using enzyme-linked immunosorbent assay to detect olanzapine compounds is to obtain a monoclonal antibody with high sensitivity to olanzapine compounds, and there is a lack of monoclonal antibodies with high sensitivity to olanzapine compounds in the prior art. SUMMARY

[0005] To this end, the technical problem to be solved by the present application is to overcome the problem of lack of a monoclonal antibody with high sensitivity to olanzapine compounds in the prior art.

[0006] To solve the above technical problem, the present application provides a hybridoma cell strain secreting a monoclonal antibody of an olanzapine compound. The monoclonal antibody secreted by the hybridoma cell strain of the present application has good sensitivity to olanzapine compounds such as olanzapine, clozapine and quetiapine, wherein the IC 50 value of olanzapine is 4.18 ng / mL, the IC 50 value of clozapine is 6.69 ng / mL, and the IC 50 value of quetiapine is 9.98 ng / mL, so that low concentrations of olanzapine compounds can be detected simultaneously.

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

[0008] Further, the hybridoma cell strain is obtained by immunizing an animal with a complete antigen prepared from a hapten, wherein the structure of the hapten is shown as formula I:

[0009]

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

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

[0012] The second object of the present application is to provide an application of the above hybridoma cell strain in detecting olanzapine compounds.

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

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

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

[0016] The fifth object of the present application is to provide a detection product of olanzapine compounds, which includes the above monoclonal antibody.

[0017] Further, the detection product further includes a coating agent.

[0018] Further, the coating agent is prepared from a hapten coupled carrier protein, wherein the carrier protein comprises bovine serum albumin.

[0019] Advantages of the present application:

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

[0021] Biological material preservation

[0022] The monoclonal cell strain MEI has been preserved in the China General Microbiological Culture Collection Center on October 31, 2024, with a preservation number of CGMCC No. 46224 and a preservation address of No. 3, Beichen West Road, Beijing Chaoyang District. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, wherein

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

[0025] The present application will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present application and implement it, but the embodiments are not limiting to the present application.

[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, magnesium sulfate anhydrous 48.84, sodium phosphate monobasic anhydrous 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L-glutamine 300, biotin 0.2, calcium D-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.

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

[0029] Carbonate buffer solution (CBS): weigh Na2CO3 1.59 g and NaHCO3 2.93 g, respectively, dissolve in a small amount of double distilled water, mix, add double distilled water to about 800 mL, mix well, adjust the pH value to 9.6, add double distilled water to 1000 mL, store at 4°C for standby.

[0030] Phosphate buffer solution (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, dissolved in 800 mL of pure water, adjusted to pH 7.2-7.4 with NaOH or HCl, and diluted to 1000 mL;

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

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

[0033] TMB developing solution: A solution: Na2HPO4·12H2O 18.43 g, citric acid 9.33 g, pure water to 1000 mL; B solution: 60 mg TMB dissolved in 100 mL ethylene glycol. A and B solutions are mixed at a ratio of 5:1 to form TMB developing solution, which is mixed before use.

[0034] The detection method involved in the following examples is as follows:

[0035] Olanzapine analogues inhibition rate detection method: the most appropriate antigen and antibody concentration in ic-ELISA was selected by chessboard test. The antigen was diluted to 0.03, 0.1, 0.3 and 1 μg / mL with carbonate buffer solution (CBS), and the antibody was diluted to 0.03, 0.1, 0.3 and 1 μg / mL with antibody diluent. After selecting the best working point, the olanzapine analogue 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, the OriginPro8.5 was used for plotting (results are shown in Figure 1 50 .

[0036] Example 1: Synthesis of Olanzapine analogue hapten

[0037] Since the olanzapine small molecule does not have immunogenicity, it cannot stimulate the immune response of mice to produce antibodies, so it is necessary to couple the olanzapine to the protein by protein coupling technology to make it immunogenic; the commonly used active groups in protein coupling technology include amino, carboxyl, hydroxyl, sulfhydryl, etc. In view of the fact that the molecular structure of olanzapine does not contain these active groups, olanzapine is derivatized.

[0038] Olanzapine 156.2 mg was dissolved in 1 mL of acetone, 122.7 mg of 6-bromohexanoic acid ethyl ester, 83 mg of anhydrous potassium carbonate, 8.3 mg of potassium iodide were added, and the mixture was refluxed at 70°C water bath 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 1M NaOH, and refluxed overnight. Then the solution was acidified to pH <2 with 2.5M HCl, and extracted with ethyl acetate three times. The prepared olanzapine analogue hapten is shown as formula I:

[0039]

[0040] Example 2: Synthesis of Olanzapine analogue complete antigen

[0041] ​Take 22 mg of olanzapine compound hapten, 6.2 mg of N-hydroxysuccinimide (NHS), dissolve in 300 μL of N,N-dimethylformamide (DMF), stir at room temperature for 15 min; then take 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 for activation. Take 6 mg of keyhole limpet hemocyanin (KLH), add to 3 mL of 0.01 M carbonate buffer solution (CBS), dissolve thoroughly, slowly add the activated hapten to the KLH dilution, 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 by ultraviolet absorption scanning method.

[0042] Example 3: Synthesis of Olanzapine Compound-Coated Original

[0043] Dissolve 13 mg of olanzapine compound hapten, 3.71 mg of N-hydroxysuccinimide (NHS) in 300 μL of anhydrous N,N-dimethylformamide (DMF), stir at room temperature for 10 min; dissolve 6.18 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in the above solution, stir at room temperature for 6-8 h for reaction to obtain a hapten activation solution; dissolve 6 mg of bovine serum albumin (BSA) in carbonate buffer solution (CBS); slowly add the hapten activation solution to the protein dilution, stir at room temperature overnight. Then dialyze the reaction solution with 0.01 M PBS solution to remove unreacted small molecules, obtain an olanzapine compound-coated original.

[0044] Example 4: Preparation of Hybridoma Cell Strains Secreting Olanzapine Compound Monoclonal Antibodies

[0045] (1) Obtaining of animal immunization

[0046] Mix and emulsify the olanzapine compound complete antigen with an equal amount of Freund's adjuvant, then subcutaneously inject the BALB / c mice at multiple points on the neck back for immunization (except for the boost immunization); the first immunization uses complete Freund's adjuvant with a dose of 100 μg per mouse; the multiple booster immunizations use incomplete Freund's adjuvant with a dose of 50 μg per mouse; the boost immunization does not use adjuvant, but is diluted with normal saline for intraperitoneal injection with a dose of 25 μg per mouse; the interval between the first immunization and the second booster immunization is one month, the interval between the multiple booster immunizations is 21 days, and the interval between the boost immunization and the last booster immunization is 18-21 days; observe the mouse immunization effect by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect the titer and inhibition of the mouse serum;

[0047] (2) Cell fusion

[0048] After sprinting immunization for three days, the cell fusion was carried out according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, and the specific steps were as follows:

[0049] a. Tail bleeding, immediately after the mice were sacrificed by cervical dislocation, were disinfected in 75% alcohol for about 5 min, and the spleen of the mice was taken out by sterile operation, grinded with the rubber head of a syringe, and passed through a 200 mesh cell screen to obtain a spleen cell suspension, which was collected, centrifuged (1200 rpm, 8 min), and washed with RPMI-1640 medium three times. After the last centrifugation, the spleen cells were diluted to a certain volume, counted, and reserved;

[0050] b. Collection of SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were expanded in 10% FBS (fetal bovine serum) RPMI-1640 medium in a 5% CO2 incubator, and the number of SP2 / 0 tumor cells was required to reach 1-4×10 7 , to ensure that the SP2 / 0 tumor cells were in the logarithmic growth phase before fusion. At the time of fusion, the tumor cells were collected and suspended in RPMI-1640 base culture solution for cell counting;

[0051] c. Fusion process 7 min: 1 mL of PEG 4000 was added to the cells from slow to fast at the first minute; at the second minute, it was left standing; at the third and fourth minutes, 1 mL of RPMI-1640 medium was added within 1 min; at the fifth and sixth minutes, 2 mL of RPMI-1640 medium was added within 1 min; at the seventh minute, 1 mL of RPMI-1640 medium was added every 10 s. Except for the second minute, the solution was constantly shaken at other times. Then it was incubated at 37°C for 5 min; centrifuged (800 rpm, 8 min), the supernatant was discarded, and it was resuspended in RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50×HAT, added to a 96-well cell plate at 200 μL / well, and cultured in a 37°C, 5% CO2 incubator.

[0052] (3) Cell screening and cell strain establishment

[0053] On the third day after cell fusion, the fusion cells were subjected to RPMI-1640 screening medium semi-replacement, and on the fifth day, they were subjected to full replacement 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.

[0054] The screening was divided into two steps: first, positive cell wells were screened by ic-ELISA method, and second, olanzapine was used as a standard for ic-ELISA method to determine the inhibitory effect of positive cells.

[0055] The cell hole with better inhibition to the olanzapine standard sample is selected, and the subcloning is performed by using the limited dilution method. Seven days later, the same method is used for detection.

[0056] The subcloning is performed at least three times according to the above method, and finally the olanzapine compound monoclonal antibody cell strain is obtained.

[0057] Example 5: Preparation and identification of olanzapine compound monoclonal antibody

[0058] Take 8-10 weeks old BALB / c mice, and inject 1 mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, inject 1x10 6 Olanzapine hybridoma cells, starting from the seventh day, collect ascites, and purify the antibody by octanoic acid-saturated ammonium sulfate method.

[0059] Under the condition of partial acid, n-octanoic acid can precipitate other impurities in the ascites except IgG immunoglobulin, and then centrifugation is performed, and the precipitate is discarded; then, the IgG type monoclonal antibody is precipitated by using an equal amount of saturated ammonium sulfate solution, centrifugation is performed, and the supernatant is discarded; after being dissolved by using 0.01M PBS solution (pH 7.4), dialysis is performed to remove salt, and finally the purified monoclonal antibody is obtained and stored at -20℃.

[0060] Using indirect competitive ELISA, the IC 50 value of the olanzapine compound monoclonal antibody to olanzapine is 4.18 ng / mL, the IC 50 value of the chlorpromazine is 6.69 ng / mL, and the IC 50 value of the quetiapine is 9.98 ng / mL, which indicates that the olanzapine compound has good sensitivity and can be used for immune analysis and detection of the olanzapine compound.

[0061] Example 6: Application of olanzapine compound monoclonal antibody

[0062] Taking olanzapine as an example, the monoclonal antibody prepared by the hybridoma cell strain through ascites in vivo is applied to the ELISA recovery test of olanzapine, and the specific steps are as follows:

[0063] The coating original diluted by the carbonate buffer solution (CBS) to a concentration of 0.1 μg / mL is coated in a 96-well enzyme-labeled plate at 100 μL per well, 37℃ is baked for 2 h, and then the plate is washed three times by using PBST at 200 μL per well, 3 min each time, and then it is patted dry;

[0064] The CBS containing 0.2% gelatin is used for blocking at 200 μL per well, 37℃ is baked for 2 h, the plate is washed three times by using PBST at 200 μL per well, 3 min each time, and then it is patted dry;

[0065] The Olanzapine standard solution of 0, 0.037, 0.111, 0.333, 1, 3, 9 and 27 ng / mL respectively was prepared with PBS, and the standard solution and the sample extraction liquid to be detected were added into the blocked enzyme label plate, 50 μL per hole, 3 holes repeated for each sample, then 50 μL of Olanzapine monoclonal antibody diluted to 0.1 μg / mL was added per hole, 37°C reaction for 30 min, then the plate was washed and dried;

[0066] 100 μL of HRP labeled goat anti-mouse IgG secondary antibody diluted to 1:3000 with PBS containing 0.1% gelatin was added per hole, 37°C reaction for 30 min, then the plate was washed and dried;

[0067] 100 μL of TMB color developing liquid was added per hole, 37°C color development for 15 min, then 50 μL of 2M H2SO4 stop solution was added per hole, and the absorbance value was measured at 450 nm;

[0068] Recovery and sample pretreatment:

[0069] Urine was selected as the detection sample.

[0070] Olanzapine standard of 0.5 ppb, 5 ppb and 50 ppb respectively was added to the sample (according to the antibody linear range and IC 50 Set the added concentration, and mix on the electric oscillator. The indirect competitive ELISA was used for the recovery test, and the recovery rates were 109%, 89% and 113% respectively.

[0071] Obviously, the above examples are only examples for clear illustration, and are not limited to the embodiments. Other different forms of changes or variations can be made by those skilled in the art on the basis of the above description. All the embodiments do not need to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A hybridoma cell line, characterized in that, The hybridoma cell strain is preserved in the China General Microbiological Culture Collection Center on October 31, 2024, and the address is No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 46224.

2. The hybridoma cell line according to claim 1, characterized in that, The hybridoma cell strain is obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structure of the hapten is shown as Formula I: 。 3. The hybridoma cell line according to claim 2, characterized in that, The complete antigen is obtained by coupling the hapten with a carrier protein.

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

5. A monoclonal antibody secreted by the hybridoma cell strain of any one of claims 1-4.

6. Use of the monoclonal antibody according to claim 5 for the detection of olanzapine-like compounds, characterized in that, The olanzapine-like compound is olanzapine, clozapine and quetiapine.

7. A detection product of an olanzapine-like compound, characterized by The detection product includes the monoclonal antibody of claim 5, and the olanzapine-like compound is olanzapine, clozapine and quetiapine.

8. The test product of claim 7, wherein, The detection product further includes a coating antigen.

9. The detection product of claim 8, wherein, The coating antigen is prepared by coupling a hapten with a carrier protein, wherein the carrier protein includes bovine serum albumin.

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