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

Abstract

The invention relates to a hybridoma cell strain secreting an etiazepine monoclonal antibody and application of the hybridoma cell strain. The hybridoma cell strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on April 18, 2024, the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.45922. The monoclonal antibody secreted by the hybridoma cell strain disclosed by the invention has good sensitivity and specificity on etiazepine. According to the present invention, the IC50 value of the etiazepine is 0.4458 ng / mL, and the etiazepine has no cross reaction with other anesthetic and psychotropic drugs (fentanyl, carfentanyl, etomidate, dexmedetomidine, dezocine and methylenedioxymethylamphetamine), such that the etiazepine can be efficiently and rapidly detected.

Description

Technical Field

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

[0002] The common name of etizidine in English is Isotonitazene, the chemical name is N, N-diethyl-2-[2-(4-ethylpropoxyphenyl)-5-nitro-1H-benzimidazol-1-yl]-1-ethylamine, the molecular weight is 410.51, and the chemical formula is C 23 H 30 N4O3, the drug was synthesized by pharmaceutical companies in the 1950s as a potential benzimidazole opioid analgesic, with an analgesic potency of about 500 times that of morphine and 20 times that of fentanyl. Currently, etizidine is regulated in the international "Single Convention on Narcotic Drugs of 1961" and my country's "Catalogue of Narcotic and Psychotropic Drugs". According to data from the US Centers for Disease Control and Prevention, in 2021, another subclass of opioids, etizidine, appeared in different regions of the United States. The popularity and strong efficacy of this type of drug led to a surge in drug overdose deaths that year.

[0003] At present, there is no clear industry testing standard for etizidine in China. In the actual in vitro testing process, gas chromatography-mass spectrometry, liquid chromatography quadrupole time-of-flight mass spectrometry, etc. are used. Although the chromatography-based method has high sensitivity and specificity, it also has some disadvantages, such as the need for thorough sample cleanup, high solvent consumption, expensive equipment, and skilled technicians. Therefore, a rapid and simple method for detecting etizidine 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 etizidine is to obtain a monoclonal antibody with high specificity and high sensitivity to etizidine, but there is no monoclonal antibody with high specificity and high sensitivity to etizidine in the prior art. 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 specificity and high sensitivity to etizidine.

[0006] In order to solve the above technical problems, the present invention provides a hybridoma cell line secreting etizidine monoclonal antibody and its application. The monoclonal antibody secreted by the hybridoma cell line of the present invention has good sensitivity and specificity to etizidine, wherein the IC 50 The value is 0.4458 ng / mL, and there is no cross reaction to fentanyl, carfentanil, etomidate, dexmedetomidine, dezocine, and methylenedioxymethamphetamine, so it can detect etizazepam efficiently and quickly.

[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 Microbiological Culture Collection Administration on April 18, 2024, with the recommended classification name as monoclonal cell strain, the deposit number is CGMCC No.45922, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] Furthermore, the hybridoma cell line is obtained by immunizing an animal with an immunogen obtained by coupling etizidine hapten with a carrier protein, wherein the structural formula of the etizidine hapten is as shown in Formula I:

[0009]

[0010] Furthermore, the immunogen is obtained by coupling the hapten to a carrier protein, and the structural formula of the immunogen is shown in Formula II.

[0011]

[0012] Furthermore, the carrier protein includes keyhole limpet hemocyanin.

[0013] Furthermore, the animal includes a mouse.

[0014] The second object of the present invention is to provide a use of the hybridoma cell line in detecting etizazepam.

[0015] The third object of the present invention is to provide a monoclonal antibody secreted by the hybridoma cell line.

[0016] The fourth object of the present invention is to provide a use of the above monoclonal antibody in detecting etizazepine.

[0017] The fifth object of the present invention is to provide an etizidine detection product, which comprises the above-mentioned monoclonal antibody.

[0018] Furthermore, the etizide detection product also includes a coating source.

[0019] Furthermore, the coating agent is prepared by coupling the etizidine hapten with a carrier protein; the carrier protein includes chicken ovalbumin.

[0020] Beneficial effects of the present invention:

[0021] The present invention screened and obtained a hybridoma cell line, and the monoclonal antibody secreted by the hybridoma cell line of the present invention has good sensitivity and specificity to etizidine. Among them, the IC 50 The value is 0.4458 ng / mL, and there is no cross reaction to fentanyl, carfentanil, etomidate, dexmedetomidine, dezocine, and methylenedioxymethamphetamine, so it can detect etizazepam efficiently and quickly.

[0022] Biomaterial Deposit

[0023] Monoclonal cell line TKLN, monoclonal cell line TKLN was deposited in the General Microbiology Center of China Culture Collection Administration on April 18, 2024, with the deposit number CGMCC No.45922, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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

[0025] Figure 1 is the standard inhibition curve of etizide monoclonal antibody. DETAILED DESCRIPTION

[0026] 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.

[0027] The culture medium involved in the following examples is as follows:

[0028] 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.

[0029] The reagents involved in the following examples are as follows:

[0030] 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.

[0031] 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;

[0032] PBST: PBS containing 0.05% Tween 20;

[0033] Antibody diluent: PBS containing 0.1% gelatin;

[0034] 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.

[0035] The detection methods involved in the following embodiments are as follows:

[0036] Method for detecting the inhibition rate of etizide: The most suitable antigen and antibody concentrations in ic-ELISA were selected by chessboard test. The antigen was diluted to 0.01, 0.03, 0.1 and 0.3 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 etizide standard was diluted to 8 concentrations (0, 0.027, 0.082, 0.247, 0.741, 2.222, 6.667 and 20 ng / mL), and the ic-ELISA operation steps were followed. Finally, OriginPro2021 was used to make a graph (the results are shown in Figure 2). Figure 1 As shown), obtain the standard inhibition curve of etizide and calculate IC 50 .

[0037] Example 1: Synthesis of etizidine hapten

[0038] Since the small molecule of etizide is not immunogenic and cannot stimulate mice to produce an immune response and then produce antibodies, etizide 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 etizide does not contain these active groups, etizide is derivatized.

[0039] Dissolve 233 mg of sodium chloroacetate in 4 mL of water to obtain liquid A; dissolve 1.382 g of anhydrous sodium carbonate in 2 mL of water to obtain liquid B; take 737.29 mg of etizidine analogs and dissolve them in 20 mL of dioxane, stir and obtain liquid C; first slowly drip liquid A into liquid C, then slowly drip liquid B into the mixed solution of A and C, and stir in a water bath at 70°C for 9 hours. Filter the reactants, rotary evaporate the filtrate to dryness, dissolve the filter residue in a small amount of boiling chloroform, filter out the insoluble matter, and purify the filtrate by silica gel column chromatography (PE / EA=5 / 1) to obtain a yellow oily product, and obtain the etizidine hapten shown in formula I, and the structural formula of the etizidine analog is shown below.

[0040]

[0041] Example 2: Synthesis of etizide complete antigen

[0042] Weigh 11.77 mg of etizidine hapten and 8.63 mg of N-hydroxysuccinimide (NHS), dissolve in 200 μL of N, N-dimethylformamide (DMF), stir at room temperature for 10 minutes; then weigh 14.72 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), add to the etizidine hapten solution, stir at room temperature for 6-8 hours to activate. Take 6 mg of keyhole limpet hemocyanin (KLH), add to 3 mL of 0.01 M carbonate buffer (CBS), fully dissolve, slowly add the activated hapten to the diluent in which KLH is dissolved, and stir at room temperature overnight. Then dialyze with 0.01 M PBS solution to remove unreacted small molecules, obtain a relatively pure complete antigen, and identify it by ultraviolet absorption scanning method.

[0043] Example 3: Synthesis of Etiazem Coating

[0044] 6.28 mg of etizidine hapten and 4.7 mg of N-hydroxysuccinimide (NHS) were dissolved in 200 μL of anhydrous N, N-dimethylformamide (DMF) and stirred at room temperature for 10 min; 8.24 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in the above solution and stirred at room temperature for 6-8 h to obtain a hapten activation solution; 6 mg of chicken ovalbumin (OVA) was dissolved in carbonate buffer (CBS); the hapten activation solution was slowly added to the protein diluent and stirred at room temperature overnight. Then, the reaction solution was dialyzed with 0.01 M PBS solution to remove unreacted small molecules to obtain the coating source.

[0045] Example 4: Preparation of hybridoma cell lines secreting etizidine monoclonal antibodies

[0046] 1. Acquisition of animal immunity

[0047] After the complete etizide antigen was mixed and emulsified with an equal amount of Freund's adjuvant, BALB / c mice were immunized by multiple subcutaneous injections at the back of the neck (except for sprint immunization); the first immunization used complete Freund's adjuvant, the dose was 100 μg / mouse; multiple booster immunizations used incomplete Freund's adjuvant and the dose was halved to 50 μg / mouse; no adjuvant was used for sprint immunization, and it was directly diluted with physiological saline and injected intraperitoneally, and the dose was halved again to 25 μg / mouse; the first immunization and the second booster immunization were separated by one month, the multiple booster immunizations were separated by 21 days, and the sprint immunization and the last booster immunization were separated by 18-21 days; the immune effect of mice was observed by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), that is, the titer and inhibition of mouse serum were detected;

[0048] 2. Cell fusion

[0049] Three days after the sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 1500) 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 is divided into two steps: the first step is to screen out the positive cell wells by the ic-ELISA method, and the second step is to use etizidine 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 etizide standard were selected and subcloned by limiting dilution method, and then tested by the same method seven days later.

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

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

[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 For the etizidine hybridoma cells, ascites was collected from the seventh day and the antibodies were purified 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] IC of monoclonal antibody against etizide was determined using indirect competitive ELISA 50 The value was 0.4458 ng / mL, and its IC value for anesthetic and psychotropic drugs was verified. 50 The cross-reaction rate to anesthetic and psychotropic analogs is less than 1%, where the cross-reaction rate = (IC 50 / Analog IC 50 )×100%. According to the cross-reaction rate value, it is shown that the antibody has good sensitivity and specificity to etizidine and can be used for etizidine immunoassay detection, as shown in Table 1.

[0062] Table 1 shows the IC values ​​of monoclonal antibodies against fentanyl, carfentanil, etomidate, dexmedetomidine, dezocine, and methylenedioxymethamphetamine. 50 And cross-reaction rate:

[0063] Table 1 Specificity results of monoclonal antibodies

[0064] <![CDATA[IC 50 (ng / mL)]]> Cross-reaction rate (%) Etiazepine 0.4458 100 Fentanyl >100 <1 Carfentanil >100 <1 Etomidate >100 <1 Dexmedetomidine >100 <1 Dezocine >100 <1 MDMA >100 <1

[0065] Example 6: Application of etizide monoclonal antibody

[0066] The monoclonal antibody prepared by hybridoma cell line through ascites in vivo was applied to the ELISA addition recovery test of etizazepine. The specific steps are as follows:

[0067] (1) Coat a 96-well ELISA plate with 0.1 μg / mL of the coating agent diluted with carbonate buffer (CBS), 100 μL per well, bake at 37°C for 2 h, wash the plate three times with PBST, 200 μL per well each time, 3 min each time, and pat dry;

[0068] (2) Block with CBS containing 0.2% gelatin, 200 μL per well, bake at 37°C for 2 h, wash the plate three times with PBST, 200 μL per well each time, 3 min each time, and pat dry;

[0069] (3) Prepare 0 ng / mL, 0.222 ng / mL, 0.666 ng / mL, 2 ng / mL, 6 ng / mL, 18 ng / mL, 54 ng / mL and 162 ng / mL of etizidine standard solutions using phosphate buffered saline (PBS), add the standard solutions and the sample extracts to be tested to the blocked ELISA plate, 50 μL per well, repeat 3 wells for each sample, then add 50 μL of etizidine monoclonal antibody diluted to 0.1 μg / mL to each well, react at 37°C for 30 min, wash the plate and pat dry;

[0070] (4) Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 in PBS containing 0.1% gelatin to each well, react at 37°C for 30 min, wash the plate and pat dry;

[0071] (5) Add 100 μL of TMB colorimetric solution to each well. After color development at 37°C for 15 min, add 50 μL of 2 mol / L H2SO4 stop solution to each well and measure the absorbance at 450 nm.

[0072] (6) Addition recovery and sample pretreatment:

[0073] Blood and urine were selected as test samples.

[0074] 0.2 ppb, 0.5 ppb, and 1 ppb of etizidine standard were added to blank blood and urine samples, respectively (according to the linear range of the antibody and IC 50 Set the concentration of addition) and then process it. Urine samples were filtered through a 0.22μm filter and directly diluted 10 times with PBS. When processing blood samples, first use 2 volumes of acetonitrile to remove proteins, then centrifuge at 12000rpm for 5min, filter the supernatant with a 0.22μm filter, and finally dilute 10 times with PBS.

[0075] The recovery rates of the spike-in recovery test were 92%, 104% and 108% respectively by indirect competitive ELISA.

[0076] 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 them 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 Microbiological Culture Collection Administration on April 18, 2024. The recommended classification name is monoclonal cell strain, the deposit number is CGMCC No.45922, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The hybridoma cell line according to claim 1, characterized in that The hybridoma cell line is obtained by immunizing an animal with an immunogen obtained by coupling etizidine hapten with a carrier protein, wherein the structural formula of the etizidine hapten is as shown in Formula I:

3. The hybridoma cell line according to claim 2, characterized in that The carrier protein includes keyhole limpet hemocyanin.

4. The hybridoma cell line according to claim 2, characterized in that The animals include mice.

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

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 etizazepine.

8. An etizidine detection product, characterized in that: The etizidine detection product comprises the monoclonal antibody according to claim 6.

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

10. The etizide detection product according to claim 9, characterized in that: The coating is prepared by coupling the etizidine hapten with a carrier protein; the carrier protein includes chicken ovalbumin.

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