Hybridoma cell strain secreting etifenin monoclonal antibody and its application

Abstract

The present application relates to a hybridoma cell strain secreting etizolam monoclonal antibody and application thereof. The hybridoma cell strain of the present application was preserved in China General Microbiological Culture Collection Center on April 18, 2024, the address of the preservation center is No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO. 45922. The monoclonal antibody secreted by the hybridoma cell strain of the present application has good sensitivity and specificity to etizolam. The IC 50 value of etizolam is 0.4458 ng / mL, and there is no cross reaction to other anesthetic and psychotropic drugs (fentanyl, carfentanyl, etomidate, dexmedetomidine, dezocine, methylenedioxymethamphetamine), so that etizolam can be efficiently and rapidly detected.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay, in particular to a hybridoma cell strain secreting etizolam monoclonal antibody and application thereof. BACKGROUND

[0002] The English generic name of etizolam 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, the chemical formula is C 23 H 30 N4O3, which was synthesized by a pharmaceutical company in the 1950s as a potential benzimidazole opioid analgesic, its analgesic potency is about 500 times that of morphine and 20 times that of fentanyl. At present, etizolam is listed in the International 1961 Narcotic Single Convention and the Narcotic and Psychotropic Drug Variety Catalog of China. According to the data of the U.S. Centers for Disease Control and Prevention, another subclass of opioid drugs, etizolam, appeared in different regions of the United States in 2021, and the prevalence of this drug and its strong potency led to a sharp increase in drug overdose deaths that year.

[0003] At present, there is no clear industry detection standard for etizolam in China, and gas chromatography-mass spectrometry, liquid chromatography-quadrupole time-of-flight mass spectrometry and other methods are used in actual in vitro detection. Although the chromatography-based method has high sensitivity and specificity, it also has 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 etizolam residues is needed.

[0004] Enzyme-linked immunosorbent assay (ELISA) is a highly 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, so it has been widely used in drug residue analysis. The premise of using enzyme-linked immunosorbent assay to detect etizolam is to obtain a monoclonal antibody with high specificity and high sensitivity to etizolam, and there is no monoclonal antibody with high specificity and high sensitivity to etizolam in the prior art. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to overcome the lack of monoclonal antibodies with high specificity and high sensitivity to etizolam in the prior art.

[0006] To solve the above technical problems, the application provides a hybridoma cell strain secreting a monoclonal antibody of etifenin and an application thereof. 50 The monoclonal antibody secreted by the hybridoma cell strain of the application has good sensitivity and specificity to etifenin, wherein the IC value of etifenin is 0.4458 ng / mL, and there is no cross reaction to fentanyl, carfentanyl, etomidate, dexmedetomidine, dezocine and methylenedioxymethamphetamine, so that etifenin can be detected efficiently and rapidly.

[0007] The first object of the application is to provide a hybridoma cell strain, which is preserved in the China General Microbiological Culture Collection Center on April 18, 2024, and the recommended classification name is a monoclonal cell strain, the preservation number is CGMCC No. 45922, and the preservation address is No. 3, Beichen West Road, Beijing City, Chaoyang District.

[0008] Further, the hybridoma cell strain is obtained by immunizing animals with an immunogen obtained after coupling of an etifenin hapten with a carrier protein, wherein the structure of the etifenin hapten is shown as formula I.

[0009]

[0010] Further, the immunogen is obtained by coupling the hapten with the carrier protein, and the structure of the immunogen is shown as formula II.

[0011]

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

[0013] Further, the animals include mice.

[0014] The second object of the application is to provide an application of the above hybridoma cell strain in detecting etifenin.

[0015] The third object of the application is to provide a monoclonal antibody secreted by the above hybridoma cell strain.

[0016] The fourth object of the application is to provide an application of the above monoclonal antibody in detecting etifenin.

[0017] The fifth object of the application is to provide an etifenin detection product, which includes the above monoclonal antibody.

[0018] Further, the etifenin detection product further includes a coating agent.

[0019] Further, the coating agent is prepared from the etifenin hapten coupled carrier protein; the carrier protein includes chicken egg white albumin.

[0020] Advantages of the present application:

[0021] The present application screens a hybridoma cell strain, the monoclonal antibody secreted by the hybridoma cell strain of the present application has good sensitivity and specificity to etifenin. The IC 50 value of etifenin is 0.4458 ng / mL, and there is no cross reaction to fentanyl, carfentanyl, etomidate, dexmedetomidine, dezocine, methylenedioxymethamphetamine, so that etifenin can be efficiently and rapidly detected.

[0022] Biological material preservation

[0023] The monoclonal cell strain TKLN is preserved in China General Microbiological Culture Collection Center on April 18, 2024, the preservation number is CGMCC No.45922, and the preservation address is No.3, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF DRAWINGS

[0024] 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 combination with the drawings, in which

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

[0026] The present application will be further described below in combination 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.

[0027] The culture medium involved in the following embodiments 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, 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.

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

[0030] Carbonate buffer solution (CBS): weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, 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 use.

[0031] Phosphate buffer solution (PBS): 8.00 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4, 2.9 g of 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;

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

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

[0034] TMB developing solution: A solution: 18.43 g of Na2HPO4·12H2O, 9.33 g of citric acid, and pure water to 1000 mL; B solution: 60 mg of TMB dissolved in 100 mL of ethylene glycol. A and B solutions are mixed at a ratio of 5:1 to obtain TMB developing solution, which is mixed before use.

[0035] The detection method involved in the following examples is as follows:

[0036] Detection method of inhibition rate of etilevodopa: The most appropriate antigen and antibody concentration in ic-ELISA was selected by chessboard test. The antigen was diluted to 0.01, 0.03, 0.1 and 0.3 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, etilevodopa 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 for plotting (results are shown in Figure 1 50 .

[0037] Example 1: Synthesis of etilevodopa hapten

[0038] Since the etilevodopa small molecule does not have immunogenicity and cannot stimulate the immune response of mice to produce antibodies, it is necessary to couple etilevodopa to proteins through protein coupling technology to make it immunogenic; the commonly used active groups in protein coupling technology include amino, carboxyl, hydroxyl, and sulfhydryl groups. In view of the fact that the molecular structure of etilevodopa does not contain these active groups, etilevodopa is derivatized.

[0039] 233 mg of sodium chloroacetate was dissolved in 4 mL of water to obtain solution A; 1.382 g of anhydrous sodium carbonate was dissolved in 2 mL of water to obtain solution B; 737.29 mg of etilevodopa analog was dissolved in 20 mL of dioxane, and after stirring, solution C was obtained; solution A was slowly added to solution C, and then solution B was slowly added to the mixture of solutions A and C, and stirring was performed at 70°C for 9 h. The reaction was filtered, the filtrate was rotary evaporated and dried, the filter residue was dissolved in a small amount of boiling chloroform, the insoluble matter was filtered out, and the filtrate was purified by silica gel column chromatography (PE / EA = 5 / 1) to obtain a yellow oily product, which was etilevodopa hapten as shown in formula I, and the structural formula of the etilevodopa analog is as follows.

[0040]

[0041] Example 2: Synthesis of etilevodopa complete antigen

[0042] ​Take 11.77 mg of etizolium hapten, 8.63 mg of N-hydroxysuccinimide (NHS), dissolve in 200 μL of N, N-dimethylformamide (DMF), stir at room temperature for 10 min; then take 14.72 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), add to the etizolium 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.

[0043] Example 3: Synthesis of etizolium coated antigen

[0044] Dissolve 6.28 mg of etizolium hapten, 4.7 mg of N-hydroxysuccinimide (NHS) in 200 μL of anhydrous N, N-dimethylformamide (DMF), stir at room temperature for 10 min; dissolve 8.24 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 chicken ovalbumin (OVA) 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 to obtain the coated antigen.

[0045] Example 4: Preparation of hybridoma cell strain secreting etizolium monoclonal antibody

[0046] 1. Obtaining of animal immunization

[0047] Mix and emulsify the etizolium complete antigen with an equal amount of Freund's adjuvant, then subcutaneously inject the BALB / c mice at multiple points on the neck and back (except for the booster 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 booster 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 booster immunization and the last booster immunization is 18-21 days; observe the immunization effect of the mice by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect the titer and inhibition of the mouse serum;

[0048] 2. Cell fusion

[0049] After sprinting immunization for three days, the cell fusion was carried out according to the conventional PEG (polyethylene glycol, molecular weight 1500) method, and the specific steps were as follows:

[0050] a. Tail bleeding, after the mice were killed by cervical dislocation, they were immediately 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 obtained a spleen cell suspension through a 200 mesh cell screen, collected, centrifuged (1200 rpm, 8 min), and the spleen cells were washed three times with RPMI-1640 medium, and finally diluted to a certain volume, counted, and reserved;

[0051] b. Collection of SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were amplified with 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, and the tumor cells were collected and suspended in RPMI-1640 basic culture solution for cell counting;

[0052] c. Fusion process 7 min: 1 mL of PEG 4000 was added to the cells from slow to fast in the first minute; 2 min, stand; 3 min and 4 min, 1 mL of RPMI-1640 medium was added within 1 min; 5 min and 6 min, 2 mL of RPMI-1640 medium was added within 1 min; 7 min, 1 mL of RPMI-1640 medium was added every 10 s. Except for the 2nd min, the solution was constantly shaken at other times. Then 37℃ incubation for 5 min; centrifugation (800 rpm, 8 min), discard the supernatant, resuspend in RPMI-1640 screening medium containing 20% fetal bovine serum, 2% 50×HAT, add 200 μL / well to a 96-well cell plate, and incubate in a 37℃, 5% CO2 incubator.

[0053] 3. Cell screening and cell strain establishment

[0054] On the 3rd day after cell fusion, the fusion cells were subjected to RPMI-1640 screening medium semi-replacement, and on the 5th day, the cells were subjected to full replacement with RPMI-1640 transition medium containing 20% fetal bovine serum and 1% 100×HT, and on the 7th day, the cell supernatant was taken for screening.

[0055] The screening was divided into two steps: first, positive cell wells were screened by ic-ELISA method, and second, the inhibitory effect of positive cells was determined by ic-ELISA method using etizolium as a standard.

[0056] The cell wells with good inhibition to the etomidate standard were selected, and subcloned by limiting dilution method. Seven days later, the same method was used for detection.

[0057] The subcloning was performed at least three times by the above method, and finally the etomidate monoclonal antibody cell strain was obtained.

[0058] Example 5: Preparation and identification of etomidate monoclonal antibody

[0059] 8-10 weeks old BALB / c mice were taken, and each mouse was injected with 1 mL of sterile paraffin oil intraperitoneally; 7 days later, each mouse was injected with 1 x 10 6 The etomidate hybridoma cells were collected from the ascites from the seventh day, and the ascites was subjected to antibody purification by caprylic acid-saturated ammonium sulfate method.

[0060] Under the condition of partial acid, n-octanoic acid can precipitate other impurities in the ascites except IgG immunoglobulin, and then centrifuged and discarded the precipitate; then the IgG type monoclonal antibody was precipitated by an equal amount of saturated ammonium sulfate solution, centrifuged and discarded the supernatant, and then dissolved in 0.01M PBS solution (pH 7.4), dialyzed and desalted, and finally obtained the purified monoclonal antibody stored at -20℃.

[0061] Using indirect competitive ELISA, the IC 50 value of the etomidate monoclonal antibody was 0.4458 ng / mL, and the IC 50 and cross-reactivity of the antibody to narcotic and psychotropic drugs were verified, and the cross-reactivity of the antibody to narcotic and psychotropic drug analogs was less than 1%, wherein the cross-reactivity = (IC 50 of etomidate / IC 50 ) x 100%, according to the cross-reactivity value, the antibody has good sensitivity and specificity to etomidate, and can be used for etomidate immunoassay detection, as shown in Table 1.

[0062] Table 1 shows the IC 50 and cross-reactivity of the monoclonal antibody to fentanyl, carfentanyl, etomidate, dexmedetomidine, dezocine, and methylenedioxymethamphetamine:

[0063] Table 1 shows the specificity results of the monoclonal antibody

[0064] IC 50 (ng / mL) Cross-reactivity (%) Eptazocine 0.4458 100 Fentanyl >100 <1 Carfentanyl >100 <1 Etomidate >100 <1 Dexmedetomidine >100 <1 Diazoxide >100 <1 Methylenedioxymethamphetamine >100 <1

[0065] Example 6: Application of etomidate monoclonal antibody

[0066] The monoclonal antibody prepared by the hybridoma cell strain through ascites in vivo was applied to the ELISA added recovery test of etomidate, and the specific steps were as follows:

[0067] (1) Coated 96-well enzyme-labeled plate with 100 μL of 0.1 μg / mL etilevodopa standard solution diluted with carbonate buffer solution (CBS), 37°C for 2 hours, then washed the plate three times with 200 μL of PBST per well, 3 minutes each time, and dried;

[0068] (2) Blocked with 200 μL of 0.2% gelatin-containing CBS per well, 37°C for 2 hours, then washed the plate three times with 200 μL of PBST per well, 3 minutes each time, and dried;

[0069] (3) Prepared 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 etilevodopa standard solutions with phosphate buffer solution (PBS), respectively, then added the standard solutions and sample extracts to be detected to the blocked enzyme-labeled plate, 50 μL per well, 3 wells for each sample, and then added 50 μL of etilevodopa monoclonal antibody diluted to 0.1 μg / mL per well, 37°C for 30 minutes, then washed and dried the plate;

[0070] (4) Added 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted to 1:3000 with 0.1% gelatin-containing PBS per well, 37°C for 30 minutes, then washed and dried the plate;

[0071] (5) Added 100 μL of TMB color developing solution per well, 37°C for 15 minutes, then added 50 μL of 2 mol / L H2SO4 stop solution per well, and measured the absorbance at 450 nm;

[0072] (6) Recovery and sample pretreatment:

[0073] Blood and urine were selected as the detection samples.

[0074] 0.2 ppb, 0.5 ppb and 1 ppb etilevodopa standard was added to the blank blood and urine samples, respectively (according to the linear range of the antibody and IC 50 Set the added concentration), and then the samples were treated. The urine sample was filtered through a 0.22 μm filter and then diluted 10 times with PBS. When treating the blood sample, first removed the protein with 2 times the volume of acetonitrile, then centrifuged at 12000 rpm for 5 minutes, filtered the supernatant with a 0.22 μm filter, and finally diluted 10 times with PBS.

[0075] The indirect competitive ELISA was used for the recovery test, and the recovery rates were 92%, 104% and 108%, respectively.

[0076] Obviously, the above embodiments are merely example for clearly illustrating, and are not limitation to the embodiments. For ordinary skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments are not required 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 China General Microbiological Culture Collection Center on April 18, 2024, and the suggested classification name is a monoclonal cell strain, the preservation number is CGMCC No. 45922, and the preservation address is No. 1, Xibahe West Road, Yard 3, Chaoyang District, Beijing.

2. The hybridoma cell line according to claim 1, characterized in that, The hybridoma cell strain is obtained by immunizing animals with an immunogen obtained after coupling an etilevodopa hapten to a carrier protein, wherein the etilevodopa hapten has the structural formula 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 animal includes a mouse.

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

6. Use of the monoclonal antibody according to claim 5 in detecting etifenac.

7. An etilevodopa test product, characterized in that, The etifenac detection product includes the monoclonal antibody according to claim 5.

8. The product of claim 7, wherein the product is a tablet. The etifenac detection product further includes a coating antigen.

9. The detomidine test product according to claim 8, characterized in that, The coating antigen is prepared by coupling a carrier protein to an etifenac hapten as shown in formula I; the carrier protein includes chicken egg white protein, 。

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