Haptens of synthetic cannabinoid ADB-butinaca, monoclonal antibody thereof and use thereof

Abstract

Disclosed in the present invention are haptens of synthetic cannabinoid ADB-BUTINACA, a monoclonal antibody thereof and the use thereof. The general structural formula of the haptens of the synthetic cannabinoid ADB-BUTINACA is shown as formula (I). The present invention provides a plurality of hapten compounds of a synthetic cannabinoid for preparing antibodies for the synthetic cannabinoid; in-vivo evaluation is carried out to obtain an optimal hapten design scheme H08, which is successfully applied in the preparation of the monoclonal antibody; and the purified antibody is used in an enzyme-linked immunosorbent assay and colloidal gold immunochromatography for measuring the content of one / more synthetic cannabinoids. Further disclosed in the present invention for the first time is the amino acid sequence of an ADB-BUTINACA specific monoclonal antibody.

Description

A hapten of a synthetic cannabinoid ADB-BUTINACA and its monoclonal antibody and application Technical Field

[0001] The present invention relates to a hapten of a synthetic cannabinoid ADB-BUTINACA, a monoclonal antibody thereof and applications thereof, and belongs to the technical field of preparation and application of synthetic cannabinoid compounds. Background Art

[0002] New psychoactive substances, also known as "designer drugs" or "laboratory drugs", are drug analogs obtained by criminals by modifying the chemical structure of controlled drugs in order to evade crackdowns. Among them, synthetic cannabinoids (SCs) have become the family with the most types of substances and the most serious abuse among new psychoactive substances, causing serious harm to public safety and social order. From July 1, 2021, synthetic cannabinoids were officially listed as a whole category. Because synthetic cannabinoids have complex and diverse structures and are metabolized into small molecules with different structures in the body, they bring huge challenges to drug detection.

[0003] Although methods such as liquid chromatography, high-performance liquid chromatography, gas chromatography, and high-performance liquid chromatography-mass spectrometry have been used to detect various synthetic cannabinoids, these methods require sample pretreatment and are costly and time-consuming. Immunoassays offer advantages such as strong antibody and antigen specificity, high sensitivity, simple operation, high throughput, and low cost. Highly specific monoclonal antibodies are the key to immunoassays. The preparation of high-affinity monoclonal antibodies requires the design of haptens based on the parent compound that retain chemical properties similar to the parent molecule and can elicit a strong immune response in mice after coupling to a carrier protein.

[0004] Although the patent application (application number: 2021112942757) discloses a synthetic cannabinoid hapten compound, the patent examples only show one hapten structure, and are aimed at synthetic cannabinoids with tert-leucine methyl ester as the head. In addition, only one hapten structure is synthesized and confirmed in the examples, and the immune effect of the hapten coupled to the protein in the body has not been verified experimentally, nor has the antigen been used to prepare monoclonal antibodies for synthetic cannabinoids.

[0005] Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a hapten structure that can be used to prepare monoclonal antibodies against synthetic cannabinoids, the antibodies prepared therefrom, and the application of the antibodies in establishing enzyme-linked immunosorbent assays and colloidal gold immunochromatographic assays to detect the content of one or more synthetic cannabinoids.

[0007] Technical solution: To solve the above technical problems, the present invention provides a synthetic cannabinoid hapten compound, the general structural formula of which is shown below:

[0008] Among them, Linker is a single bond, double bond, triple bond, C 1-8 Hydrocarbon, C with or without amino 1-8 Hydrocarbon groups, with or without amide bonds 1-8 a hydrocarbon group, p-phenyl, m-phenyl, o-phenyl, 1,4-divinylphenyl, vinylphenyl, ethynylphenyl, 1,4-diethynylphenyl, biphenyl, 1,4-methylenephenyl, or 9,10-anthryl.

[0009] The present invention also provides the use of the synthetic cannabinoid hapten in the preparation of a synthetic cannabinoid complete antigen.

[0010] The present invention also provides a synthetic cannabinoid complete antigen prepared from the synthetic cannabinoid hapten compound, the structure of which is shown below:

[0011] The synthetic cannabinoid complete antigen is obtained by coupling a carrier protein with a hapten, and the carrier protein includes KLH and BSA.

[0012] The present invention also provides a synthetic cannabinoid monoclonal antibody, which is prepared by immunizing animals with the synthetic cannabinoid complete antigen.

[0013] The variable region amino acid sequence of the antibody light chain is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.3; the variable region amino acid sequence of the heavy chain is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.4.

[0014] The present invention also provides the use of the synthetic cannabinoid hapten or the synthetic cannabinoid complete antigen in the preparation of cannabinoid monoclonal antibodies.

[0015] The present invention also provides the use of the synthetic cannabinoid hapten compound or the synthetic cannabinoid complete antigen or the monoclonal antibody in detecting the content of synthetic cannabinoids.

[0016] The present invention also provides a colloidal gold immunochromatographic test strip, wherein the test strip contains the cannabinoid monoclonal antibody.

[0017] The present invention also provides the use of the colloidal gold test strip in detecting synthetic cannabinoids.

[0018] The synthetic cannabinoids include one or more of ADB-BUTINACA, ADB-CHMINACA, ADB-CHMICA, AB-FUBINACA, AB-CHMINACA, ADB-3en-BUTINACA, AB-4en-PINACA, ADB-4en-PINACA, EMB-FUBINACA or MDMB-BUTINACA butanoic acid metabolite.

[0019] The present invention also provides light and heavy chain expression plasmids of the synthetic cannabinoid monoclonal antibody 2E4, and briefly describes its expression method in the HEK293F eukaryotic system.

[0020] The present invention also provides a plasmid vector containing the nucleotide sequence shown in SEQ ID NO.3 and / or the nucleotide sequence shown in SEQ ID NO.4.

[0021] The present invention also provides a method for constructing the plasmid vector, comprising the following steps: using pcDNA3.4 as an expression vector, the nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8 as constant region sequences, SEQ ID NO.5 as a signal peptide sequence, and the nucleotide sequence shown in SEQ ID NO.3 and / or the nucleotide sequence shown in SEQ ID NO.4 as a variable region sequence.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention provides a variety of synthetic cannabinoid hapten compounds for the preparation of synthetic cannabinoid antibodies. The optimal hapten design scheme H08 is obtained through in vivo evaluation and successfully applied to the preparation of monoclonal antibodies. The purified antibodies are applied to enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography to detect the content of one or more synthetic cannabinoids; 2. The present invention also reports the amino acid sequence of the ADB-BUTINACA-specific monoclonal antibody for the first time; 3. The specific monoclonal antibody is successfully expressed in a eukaryotic system. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a HPLC spectrum of synthetic cannabinoid hapten compound H01;

[0024] FIG2 is a HPLC spectrum of the synthetic cannabinoid hapten compound H02;

[0025] FIG3 is a HPLC spectrum of synthetic cannabinoid hapten compound H03;

[0026] FIG4 is a HPLC spectrum of synthetic cannabinoid hapten compound H04;

[0027] FIG5 is a HPLC spectrum of synthetic cannabinoid hapten compound H05;

[0028] FIG6 is a HPLC spectrum of the synthetic cannabinoid hapten compound H06;

[0029] FIG7 is a HPLC spectrum of synthetic cannabinoid hapten compound H07;

[0030] FIG8 is a HPLC spectrum of synthetic cannabinoid hapten compound H08;

[0031] FIG9 is a HPLC spectrum of synthetic cannabinoid hapten compound H09;

[0032] FIG10 is a HPLC spectrum of synthetic cannabinoid hapten compound H10;

[0033] Figure 11 shows the antibody titers of mice immunized 21 and 35 days after the complete synthetic cannabinoid antigen was mixed with adjuvant;

[0034] Figure 12 shows the affinity of antibodies to ADB-BUTINACA 35 days after immunization of mice with complete synthetic cannabinoid antigen mixed with adjuvant;

[0035] FIG13 shows the IC of the purified ADB-BUTINACA-specific monoclonal antibody 2E4 and ADB-BUTINACA measured by ELISA. 50 value;

[0036] FIG14 is a standard curve of ADB-BUTINACA in hair and urine determined by ELISA;

[0037] FIG15 is a colloidal gold immunochromatographic assay of ADB-BUTINACA in hair and urine: A, C, and E are colloidal gold test strips with PBS, urine, and hair as standard curves, respectively; B, D, and F are colloidal gold test strips with PBS, urine, and hair as standard curves, respectively, as concentration versus T / C curves;

[0038] Figure 16 is a schematic diagram of the construction of light and heavy chain expression vectors;

[0039] FIG17 is a validation of the activity of synthetic cannabinoid antibodies expressed in HEK 293F cells. DETAILED DESCRIPTION

[0040] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0041] Example 1 Preparation of ADB-BUTINACA hapten H01

[0042] Synthesis route:

[0043] 1. Condensation reaction

[0044] 500 mg of 1H-indazole-3-carboxylic acid, 887 mg of carbodiimide hydrochloride (EDCI), and 625 mg of 1-hydroxybenzotriazole (HOBt) were weighed and dissolved in 3 mL of N,N-dimethylformamide (DMF) and stirred under N2 protection for dissolution. The reaction solution was stirred for half an hour. 617 mg of L-tert-leucamide hydrochloride was dissolved in 5 mL of DMF and added to the reaction solution simultaneously with 1.196 g of N,N-diisopropylethylamine (DIPEA). The reaction was stirred at room temperature for 4 hours. TLC confirmed the complete reaction of the raw materials. The reaction was terminated with an appropriate amount of ethyl acetate, washed twice with saturated sodium bicarbonate and once with saturated sodium chloride, and the ethyl acetate layer was collected, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, added to silica gel powder, and passed through a silica gel chromatography column (developing solvent: petroleum ether:ethyl acetate mass ratio of 4:1) to obtain the product as a white solid powder (Intermediate 1).

[0045] 2. Substitution reaction

[0046] Intermediate 1 (500 mg, 1 eq) and NaH (87 mg, 2 eq) were dissolved in 5 mL of DMF and stirred under N2 protection and an ice bath. The reaction mixture was stirred for half an hour. Ethyl 4-bromobutyrate (457 mg, 1.5 eq) was diluted with 2 mL of DMF and added to the reaction system via a syringe. The mixture was stirred at room temperature until the reaction was complete. The reaction was terminated with ethyl acetate, and the mixture was washed once with saturated sodium bicarbonate and once with saturated sodium chloride. The ethyl acetate layer was collected, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, added with silica gel powder, and dried by spin drying. The mixture was then passed through a silica gel chromatography column (developing solvent: petroleum ether: ethyl acetate 10:1) to obtain a white solid powder (Intermediate 2).

[0047] 3. Ester hydrolysis reaction

[0048] Intermediate 2 (200 mg, 1 eq) was dissolved in 5 mL of methanol, and 100 μL of saturated KOH solution was added. The reaction was stirred at room temperature and monitored by TLC until complete reaction. Excess methanol was removed using a rotary evaporator, and an appropriate amount of pure water was added to dissolve the solid. While stirring under an ice bath, the pH of the reaction solution was adjusted to 4 with 1 M hydrochloric acid. A large amount of solid precipitated, which was filtered and then dried to obtain the ester hydrolysis product H01. The purity of the target compound H01 was determined by high-performance liquid chromatography (HPLC) using an Eclipse XDB-C18 (4.6 × 250 mm, 5 μm) column. Peak times were measured at a UV wavelength of 254 nm on an Agilent 1100 liquid chromatograph. The aqueous phase consisted of ultrapure water containing 1 / 1000 ppm formic acid, and the organic phase consisted of acetonitrile containing 1 / 1000 ppm formic acid. Gradient injection was performed from 0 min to 30 min, with the organic phase ratio increasing from 10% to 100%. The HPLC chromatogram of H01 is shown in Figure 1. 1H NMR(300MHz, DMSO-d6)δ12.16(s,1H),8.17(d,J=8.2Hz,1H),7.84–7.69(m,2H),7.59(d,J=9.7Hz,1H),7.53–7.45(m,1H),7 .35–7.24(m,2H),4.55(t,J=7.0Hz,2H),4.46(d,J=9.7Hz,1H),2.35–2.23(m,2H),2.08(m,J=7.9,7.4Hz,2H),1.00(s,9H). 13 C NMR(75MHz,DMSO-d6)δ174.30, 172.28,161.46,141.10,136.91,127.27,123.11,122.41,122.17,110.8 7,59.12,48.48,39.94,35.03,31.10,27.12,25.39.HRMS(ESI)m / z:[M+H] + calculated=361.1876; found=361.1877.

[0049] Example 2 Preparation of ADB-BUTINACA haptens H02 to H05

[0050] 1. Preparation of ADB-BUTINACA hapten H02

[0051] Synthesis route:

[0052] The hapten H02 was obtained by replacing ethyl 4-bromobutyrate in the substitution reaction in Example 1 with 1.5 eq of methyl 5-bromovalerate while keeping other conditions unchanged.

[0053] HPLC detection was performed according to the method of Example 1. The HPLC chromatogram of hapten H02 is shown in FIG2 . 1 H NMR (300MHz, DMSO-d6) δ11.99(s,1H),8.17(d,J=8.0Hz,1H),7.79(t,J=9.4Hz,2H),7.59(d,J=9.7Hz,1H),7.48(t,J=7.1Hz,1H),7.29(d, J=14.6Hz,2H),4.53(t,J=7.0Hz,2H),4.46(d,J=9.7Hz,1H),2.26(t,J=7.4Hz,2H),1.95–1.78(m,2H),1.50(p,J=7.4Hz,2H),0.99(s,9H). 13C NMR(75MHz,DMSO-d6)δ174.75,172.28,161.48,141.13,136.81,127.19,123.05,122.37 ,122.13,110.99,59.09,48.94,35.04,33.56,29.41,27.12,22.21.HRMS(ESI)m / z:[M+H] + calculated=375.2032; found=375.2026.

[0054] 2. Preparation of ADB-BUTINACA hapten H03

[0055] Synthesis route:

[0056] The ethyl 4-bromobutyrate in the substitution reaction in Example 1 was replaced with 1.5 eq of methyl 6-bromohexanoate to obtain hapten H03.

[0057] HPLC detection was performed according to the method of Example 1. The HPLC chromatogram of hapten H03 is shown in FIG3 . 1 H NMR (300MHz, DMSO-d6) δ11.99(s,1H),8.17(d,J=8.1Hz,1H),7.84–7.68(m,2H),7.59(d,J=9.7Hz,1H),7.47(t,J=7.7Hz,1H),7.35–7.21(m,2 H),4.60–4.41(m,3H),2.22(dt,J=26.0,7.3Hz,2H),1.87(p,J=7.2Hz,2H),1.55(dq,J=14.9,7.3Hz,2H),1.30(q,J=8.3Hz,2H),1.00(s,9H). 13 C NMR(75MHz,DMSO-d6)δ174.92,172.29,161.49,141.10,136.75,127.16,123.03,122.37,12 2.11,111.01,59.10,49.07,35.04,34.11,29.66,27.12,26.19,24.53.HRMS(ESI)m / z:[M+H] + calculated=389.2189; found=389.2193.

[0058] 3. Preparation of ADB-BUTINACA hapten H04

[0059] Synthesis route:

[0060] Hapten H01 (200 mg, 1 eq) prepared in Example 1 was condensed with methyl 4-aminobutyrate hydrochloride (200 mg, 1 eq) in the presence of EDCI (200 mg, 1.5 eq) and HOBt (200 mg, 1.5 eq) to produce a methyl ester-protected hapten compound. The methyl ester was then demethylated under alkaline conditions to produce the synthetic cannabinoid hapten compound H04. High-performance liquid chromatography (HPLC) analysis was performed according to the method of Example 1. The HPLC chromatogram of H04 is shown in Figure 4. 1 H NMR (300MHz, DMSO-d6) δ12.06(s,1H),8.18(d,J=8.2Hz,1H),7.87–7.72(m,3H),7.60(d,J=9.7Hz,1H),7.47(t,J=7.7Hz,1H),7.3 5–7.20(m,2H),4.61–4.38(m,3H),3.05(q,J=6.7Hz,2H),2.21(t,J=7.4Hz,2H),2.10(s,4H),1.61(p,J=7.2Hz,2H),1.00(s,9H). 13 C NMR(75MHz,DMSO-d6)δ174.70,172.32,171.60,161.51,141.13,136.90,127.21,123.06,122.43,12 2.15,110.92,59.13,48.79,38.35,35.03,32.38,31.53,27.12,25.96,25.01.HRMS(ESI)m / z:[M+H] + calculated=446.2403; found=446.2373.

[0061] 4. Preparation of ADB-BUTINACA hapten H05

[0062] Synthesis route:

[0063] The prepared hapten H03 (200 mg, 1 eq) was reacted with beta-alanine ethyl ester hydrochloride (98 mg, 1.2 eq) in the presence of EDCI (154 mg, 1.5 eq), HOBt (108 mg, 1.5 eq), and DIPEA (207 mg, 3 eq) to form a methyl ester-protected hapten compound. This methyl ester was then demethylated under alkaline conditions to produce the synthetic cannabinoid hapten compound H05. High-performance liquid chromatography (HPLC) was performed according to the method of Example 1. The HPLC chromatogram of H05 is shown in Figure 5 . 1H NMR (300MHz, DMSO-d6) δ8.32(t,J=5.5Hz,1H),8.16(d,J=8.1Hz,1H),7.79(d,J=8.6Hz,1H),7.5 8(d,J=9.7Hz,1H),7.51–7.42(m,1H),7.33–7.23(m,1H),4.58–4.42(m,3H),3.56(s,3H),3.09( ddq,J=47.1,12.9,6.4Hz,2H),2.31(t,J=7.2Hz,2H),1.84(p,J=7.2Hz,2H),1.54(dt,J=14.4,7 .1Hz,2H),1.43(dt,J=13.6,6.7Hz,2H),1.35–1.20(m,2H),0.98(s,9H),0.90(t,J=7.4Hz,3H). 13 C NMR(75MHz,DMSO-d6)δ173.48,172.37,161.51,141.12,136.78,127.21,123.06,122.35,122.1 1,110.96,59.13,48.94,35.23,35.07,35.01,34.44,29.46,27.10,22.89.HRMS(ESI)m / z:[M+H] + calculated=446.2403; found=446.2401.

[0064] Example 3 Preparation of ADB-BUTINACA haptens H06 to H08

[0065] 1. Preparation of ADB-BUTINACA hapten H06

[0066] Synthesis route:

[0067] 1H-indazole-3-carboxylic acid (500 mg, 1 eq) and L-tert-leucine methyl ester hydrochloride (673 mg, 1.2 eq) were condensed in the presence of EDCI (887 mg, 1.5 eq), HOBt (625 mg, 1.5 eq) and DIPEA (1196 mg, 3 eq) to produce intermediate 3; the intermediate 3 (500 mg, 1 eq) was reacted with 1-bromobutane (285 mg, 1.2 eq) in the presence of NaH (83 mg, 2 eq) to produce intermediate 3. A substitution reaction occurred to generate intermediate 4; intermediate 4 was hydrolyzed and demethylated under alkaline conditions to generate intermediate 5 with a carboxyl group. Intermediate 5 (200 mg, 1 eq) was condensed with ethyl 3-aminopropionate hydrochloride (111 mg, 1.2 eq) in the presence of EDCI (174 mg, 1.5 eq), HOBt (122 mg, 1.5 eq), and DIPEA (234 mg, 3 eq) to obtain a hapten precursor, which was hydrolyzed under alkaline conditions to obtain the synthetic cannabinoid hapten compound H06. High-performance liquid chromatography (HPLC) was performed according to the method of Example 1. The HPLC chromatogram of H06 is shown in Figure 6. 1 H NMR (300MHz, DMSO-d6) δ11.99(s,1H),8.17(d,J=8.0Hz,1H),7.79(t,J=9.4Hz,2H),7.59(d,J=9.7Hz,1H),7.48(t,J=7.1Hz,1H),7.2 9(m,2H),4.53(t,J=7.0Hz,2H),4.46(d,J=9.7Hz,1H),2.26(t,J=7.4Hz,2H),1.95–1.78(m,2H),1.50(p,J=7.4Hz,2H),0.99(s,9H). 13 C NMR(75MHz,DMSO-d6)δ174.62,170.17,161.46,141.13,136.66,127.15,123.03,122.36,122.10,1 10.99,59.25,48.95,38.39,35.29,31.97,31.60,27.05,24.90,19.87,13.94.HRMS(ESI)m / z:[M+H] + calculated=417.2502; found=417.2508.

[0068] 2. Preparation of ADB-BUTINACA hapten H07:

[0069] Synthetic cannabinoid hapten compound H07 was obtained by replacing 4-aminobutyric acid methyl ester hydrochloride with 5-aminovalerate methyl ester in the above-mentioned process for preparing synthetic cannabinoid hapten compound H06. The other steps and starting materials were the same. Intermediate 5 (200 mg, 1 eq) was condensed with 5-aminovalerate methyl ester (122 mg, 1.2 eq) in the presence of EDCI (174 mg, 1.5 eq), HOBt (122 mg, 1.5 eq), and DIPEA (234 mg, 3 eq) to obtain a hapten precursor, which was then hydrolyzed under alkaline conditions to obtain synthetic cannabinoid hapten compound H07. High-performance liquid chromatography (HPLC) was performed according to the method of Example 1. The HPLC chromatogram of H07 is shown in Figure 7. 1 H NMR (300MHz, DMSO-d6) δ12.13(s,1H),8.33(t,J=5.5Hz,1H),8.16(d,J=8.1Hz,1H),7.80(d,J=8.6Hz, 1H),7.58(d,J=9.7Hz,1H),7.47(ddd,J=8.4,6.9,1.0Hz,1H),7.33–7.24(m,1H),4.58–4.42(m,3H),3. 18(dq,J=12.4,6.5Hz,1H),3.00(dq,J=12.4,6.5Hz,1H),2.21(t,J=7.0Hz,2H),1.84(p,J=7.2Hz,2H), 1.49(ddd,J=17.4,13.3,6.2Hz,4H), 1.29(dq,J=14.4,7.2Hz,2H), 0.98(s,9H), 0.90(t,J=7.4Hz,3H). 13 C NMR (75MHz, DMSO-d6) δ174.84,170.04,161.44,141.13,136.67,127.15,123.03,122.36,122.11,111. 00,59.21,48.95,38.63,35.32,33.75,31.97,28.93,27.07,22.53,19.87,13.95.HRMS(ESI)m / z:[M+H] + calculated=431.2658; found=431.2660.

[0070] 3. Preparation of ADB-BUTINACA hapten H08:

[0071] Synthesis route:

[0072] Intermediate 5 (200 mg, 1 eq) was condensed with methyl 6-aminohexanoate hydrochloride (132 mg, 1.2 eq) in the presence of EDCI (174 mg, 1.5 eq), HOBt (122 mg, 1.5 eq), and DIPEA (234 mg, 3 eq) to yield a hapten precursor, which was then hydrolyzed under alkaline conditions to yield synthetic cannabinoid hapten compound H08. High-performance liquid chromatography (HPLC) was performed according to the method of Example 1. The HPLC chromatogram of H08 is shown in Figure 8 . 1 H NMR (300MHz, DMSO-d6) δ8.32(t,J=5.5Hz,1H),8.16(d,J=8.1Hz,1H),7.79(d,J=8.6Hz, 1H),7.58(d,J=9.7Hz,1H),7.53–7.41(m,1H),7.35–7.23(m,1H),4.58–4.39(m,3H),3.1 7(dq,J=12.9,6.7Hz,1H),2.98(dq,J=12.6,6.5Hz,1H),2.10(t,J=7.3Hz,2H),1.84(p, J=7.3Hz,2H),1.54–1.35(m,4H),1.34–1.21(m,4H),0.98(s,9H),0.90(t,J=7.4Hz,3H). 13 C NMR(75MHz,DMSO-d6)δ175.45,169.99,161.44,141.12,136.69,127.15,123.02,122.36,122.11,110.99, 59.21,48.95,38.85,35.33,35.19,31.97,29.21,27.07,26.71,25.07,19.87,13.94.HRMS(ESI)m / z:[M+H] + calculated=445.2815; found=445.2781.

[0073] Example 4 Preparation of ADB-BUTINACA hapten H09

[0074] Synthesis route:

[0075] Hapten H06 (200 mg, 1 eq) was condensed with methyl 4-aminobutyrate hydrochloride (89 mg, 1.2 eq) in the presence of EDCI (138 mg, 1.5 eq), HOBt (97 mg, 1.5 eq), and DIPEA (186 mg, 3 eq) to produce a hapten precursor, which was then hydrolyzed under alkaline conditions to yield the synthetic cannabinoid hapten compound H09. High-performance liquid chromatography (HPLC) was performed according to the method of Example 1. The HPLC chromatogram of H09 is shown in Figure 9. 1 H NMR (300MHz, DMSO-d6) δ12.07(s,1H),8.34(t,J=5.4Hz,1H),8.16(d,J=8.1Hz,1H),7.82(dd,J =10.4,7.1Hz,2H),7.58(d,J=9.7Hz,1H),7.47(ddd,J=8.4,6.9,1.0Hz,1H),7.35–7.23(m,1H), 4.67–4.35(m,3H),3.28–2.85(m,4H),2.20(t,J=7.4Hz,2H),2.08(t,J=7.5Hz,2H),1.84(p,J=7 .2Hz,2H),1.62(dp,J=14.3,7.3Hz,4H),1.34–1.23(m,2H),0.98(s,9H),0.90(t,J=7.4Hz,3H). 13 C NMR(75MHz,DMSO-d6)δ174.75,171.99,170.08,161.45,141.13,136.66,127.16,123.04,122.36,122.10,111.00, 59.25,48.96,38.74,38.33,35.33,33.33,31.97,31.58,27.07,25.70,25.09,19.87,13.94.HRMS(ESI)m / z:[M+H] + calculated=502.3029; found=502.2988.

[0076] Example 5 Preparation of ADB-BUTINACA hapten H10

[0077] Synthesis route:

[0078] Intermediate 4 (200 mg, 1 eq) was dissolved in 5 mL of tetrahydrofuran, and NaBH4 (65.71 mg, 3 eq) and 0.2 mL of methanol were added. The ester bond was reduced to an alcoholic hydroxyl group under heating to produce Intermediate 6. The hydroxyl group of Intermediate 6 was oxidized to an aldehyde group in CHCl under the action of PCC (pyridinium chlorochromate, 65.71 mg, 3 eq) to produce Intermediate 7. Intermediate 7 (100 mg, 1 eq), carboxymethyloxyamine hemihydrochloride (87 mg, 3 eq), and pyridine (75 mg, 3 eq) were reacted in methanol to produce hapten H10. High-performance liquid chromatography (HPLC) was performed according to the method of Example 1. The HPLC chromatogram of H10 is shown in Figure 10. 1 H NMR (300MHz, DMSO-d6) δ12.71(s,1H),8.35(d,J=9.6Hz,1H),8.16(d,J=8.1Hz,1H),7.79(dd,J=8.3,3.5Hz,2H),7.52 –7.40(m,1H),7.28(t,J=7.6Hz,1H),4.63–4.40(m,5H),1.86(p,J=7.2Hz,2H),1.35–1.23(m,2H),1.03–0.81(m,12H). 13 C NMR(75MHz,DMSO-d6)δ171.45,161.91,150.90,141.02,137.06,127.08,122.89,122.65,12 2.21,110.90,70.38,55.89,48.96,35.71,32.00,26.83,19.91,13.99.HRMS(ESI)m / z:[M+H] + calculated=389.2189; found=389.2163.

[0079] Example 6 Synthesis of Complete ADB-BUTINACA Antigen

[0080] 5 mg of hapten compounds H01-10 were weighed separately, and EDCI (6 eq), NHS (6 eq), and Et3N (6 eq) were dissolved in 500 μL DMF with the hapten compounds H01-10, respectively. The mixture was stirred at room temperature for 6 h until the hapten reaction was complete. KLH PBS solution (10 mg of KLH protein was weighed and dissolved in PBS solution to 5 mg / mL) or BSA PBS solution (10 mg of BSA protein was weighed and dissolved in PBS solution to 5 mg / mL) was added, and the mixture was stirred at 4°C overnight. The mixture was taken out the next day, placed in a MW 3000 dialysis bag, and dialyzed against 500 mL of PBS (pH 7.4) each time for a total of 4 times to obtain PBS solutions of immune antigens KLH-H01-10 and coating antigens BSA-H01-10, respectively. The PBS solutions were aliquoted and frozen at -80°C for use. Among them, the immune antigen KLH-H01~10 and the coating antigen BSA-H01~10 are both complete antigens. The structural formula of the complete antigen is as follows:

[0081] Wherein, KLH / BSA in the above structural formula represents KLH protein or BSA protein.

[0082] Example 7 ADB-BUTINACA complete antigen immunization

[0083] Six-week-old Balb / c female mice were injected subcutaneously with KLH-H01-10 immunizing antigen at multiple sites on their backs at a dose of 0.1 mL / 50 μg / mouse. For the first immunization, 100 μg of KLH-H01-10 immunizing antigen was emulsified in equal volumes with Freund's complete adjuvant (Sigma, F5581). Two weeks later, a second booster immunization was performed using an equal volume of 50 μg of KLH-H01-10 emulsified in Freund's incomplete adjuvant (Sigma, F5506). Two weeks later, a third booster immunization was performed using the same method as the second booster immunization. Blood was collected from the mice's orbits on days 21 and 35 after the initial immunization. Serum was collected and aliquoted and frozen at -80°C until use.

[0084] Example 8 Determination of serum antibody titer and affinity with ADB-BUTINACA after mouse immunization

[0085] 1. Serum titer determination:

[0086] Coating: Dilute BSA-H01-10 coating antigens (for detection of immune antigen serum, use coating antigens prepared with the corresponding haptens) to 1 μg / ml in 0.01M pH 7.4 PBS solution, add 100 μl / well to a 96-well ELISA plate, seal with film, and incubate overnight at 4°C.

[0087] Blocking: Wash the plate three times with PBST, add 100 μl / well of blocking solution, and incubate at 37°C for 1.5 h.

[0088] Incubation with primary antibody: After blocking, wash the plate 5 times with PBST, and add 50 μl of serum with a certain gradient dilution (starting from 1:1000 with PBST, 3-fold dilution, a total of 12 dilution gradients, i.e. 1:1000, 1:3000, 1:9000, 1:27000, 1:81000, 1:243000, 1:729000, 1:2187000, 1:6561000, 1:19683000, 1:59049000, 1:177147000) and blank control serum to each well;

[0089] Secondary antibody incubation: After the primary antibody incubation, the plate was washed five times with PBST, and 100 μL / well of goat anti-mouse HRP enzyme-labeled secondary antibody (Jackson ImmunoResearch, 205-035-108) diluted in PBST was added (PBST: goat anti-mouse HRP enzyme-labeled secondary antibody = 1:5000, v / v) and incubated at 37°C for 30 min.

[0090] Color development: After the secondary antibody incubation, wash the plate five times with PBST, add TMB color development solution (Biyuntian, P0209) at a volume of 50 μL / well, and incubate at 37°C in the dark for 10 min;

[0091] Termination: After color development is complete, add 50 μL / well of 2M H2SO4 to terminate the color reaction. Absorbance is measured at 450 nm using an MD i3x microplate reader. The antibody titer is determined by the OD450 value at the maximum serum dilution being 2.1 times higher than that of blank serum. The results are shown in Figure 11. Comparing antibody titers at 21 and 35 days, H04, with tail-modified linker sites, produced the highest antibody titer after immunization of mice, reaching 3×10 at 35 days. 6 The hapten H06-H10 with modified head as the linker site produced the highest antibody titer after immunizing mice. The antibody titer reached 10 at 35 days. 6 .

[0092] 2. Affinity determination with ADB-BUTINACA

[0093] The steps are the same as those for antibody titer determination, except that during the primary antibody incubation, 50 μL of serum at a certain dilution (OD value of 1) and 50 μL of different concentrations of ADB-BUTINACA (laboratory-preserved) are added and incubated together. The competitive concentrations of ADB-BUTINACA are 400, 133, 44.4, 14.8, 4.94, 1.65, 0.549, 0.183, 0.061, 0.020, 0.007, and 0 μM, and the incubation is at 37°C for 1 hour. The chemical structure of ADB-BUTINACA is as follows: The results are shown in Figure 12, comparing the 35-day IC 50 The results showed that after immunizing mice with haptens H01-H05, whose tails were modified with linker sites, the antibodies produced by H02 had the highest affinity for ADB-BUTINACA; after immunizing mice with haptens H06-H10, whose heads were modified with linker sites, the antibodies produced by H02 had the highest affinity for ADB-BUTINACA. 50 Found that H08 IC 50 The value was lower, indicating that the antibodies produced were more specific to ADB-BUTINACA, so mice immunized with H08 were subsequently selected for hybridoma fusion.

[0094] Example 9 Preparation of ADB-BUTINACA Monoclonal Antibody

[0095] Preparation of immune spleen cells: The antibody titers and specificities of the serum of mice immunized with KLH-H01~10 antigens in ten groups were evaluated by ELISA. 50 The value is the smallest (IC 50The mice in the KLH-H08 immunization group (with a KLH-H08 value of 1.044 μM) were given a final boost immunization three days before fusion, i.e., 150 μg of the immune antigen, i.e., KLH-H08, was intraperitoneally injected without adjuvant; three days later, spleen cells of the mice after the boost immunization were prepared for extraction. First, kill the mouse by dislocating the neck, soak it in 75% alcohol for 10 minutes, transfer it to a sterile culture dish on the clean bench, and fix it with its abdomen facing up; use scissors to carefully cut the mouse skin to expose the mouse abdominal cavity, disinfect the entire abdomen with alcohol cotton, cut the peritoneum, find the spleen, and carefully separate it; place the spleen in a culture dish containing 1640 culture medium, carefully remove excess fat on the spleen, and then transfer it to a clean culture dish with a small amount of 1640 culture medium; take a 5mL syringe, use the needle to pierce several small holes in the spleen, aspirate the culture medium with the syringe, penetrate the spleen from one end, and gently eject the culture medium. At this time, spleen cells can be seen released into the 1640 culture medium. Repeat the blowing until the spleen turns white. Pass the spleen cell suspension through a 70μm sieve to remove large pieces of tissue, collect the suspension, centrifuge and discard the supernatant to obtain spleen cells; count the cells.

[0096] Cultivation of SP2 / 0 myeloma cells: Thaw SP2 / 0 myeloma cells frozen in a -80°C freezer one week in advance. Preheat the water bath to 37°C. Remove the cryovial and place it directly in ultrapure water, shaking continuously until the cryopreservative is completely thawed. Centrifuge at 1000 rpm / min for 5 minutes at room temperature, discard the supernatant, resuspend the cell pellet in an appropriate amount of complete medium, transfer the pellet to a cell culture flask, add 5 mL of complete medium, and culture in a cell culture incubator (37°C, 5% CO2). Once the cells have largely adhered and grown to a density exceeding 75% of the bottom surface area of ​​the dish, gently aspirate the cells with a pipette, centrifuge, and passage into multiple cell culture flasks. Add complete medium to the flasks up to 5 mL, and continue culturing in the incubator. Change the complete medium regularly based on cell growth and cell culture medium color.

[0097] Cell fusion: SP2 / 0 myeloma cells and spleen cells were mixed at a ratio of 1:5, transferred to a 50 mL centrifuge tube, centrifuged at 1000 rpm / min for 5 minutes, discarded the supernatant, and aspirated the remaining liquid to reduce the effect on the cell fusion effect; gently tap the centrifuge tube to loosen the precipitated cells at the bottom of the centrifuge tube, and place it in a 37°C water bath; accurately draw 1 mL of 37°C preheated PEG 1450, slowly at first and then quickly, while constantly rotating the centrifuge tube to mix it evenly, add PEG 1450 dropwise to the centrifuge tube within 1 minute, and let it stand for 1 minute after the addition is complete; then add 37°C preheated 1640 culture medium dropwise at a rate of 1 mL per minute, 2 mL per minute, 3 mL per minute, and 5 mL per minute to terminate the cell fusion agent PEG. 1450, then place it in an incubator for 10 minutes, centrifuge it at 1500 rpm / min for 5 minutes, and discard the supernatant; add preheated HAT complete medium, gently pipette the deposited cells to suspend the cells, and remember not to blow hard to avoid dispersing and breaking the fused cells; mix the cell suspension and add it to a 96-well cell culture plate, 200 μL per well, and then place the cell culture plate in a cell culture incubator for culture.

[0098] Screening and monocloning of hybridomas: On the second day after cell fusion, check whether the cell fusion experiment is contaminated by observing the bottom of the cell plate under a microscope to see if there are contaminants. On the third day after fusion, perform a half-change of the medium for the cells using HAT complete medium. On the 5th to 7th day after fusion, perform a half-change of the medium for the cells using HT complete medium according to the cell growth status. The medium change method is the same as on the third day, that is, remove half of the original culture medium and add an equal amount of fresh culture medium. After 7 days, perform a full medium change of the cells to avoid the influence of specific antibodies secreted by unfused spleen cells on the test results. On the second day after full medium change, take a small amount of cell culture supernatant, dilute it, and use indirect ELISA to screen the positive wells. Add 400nM target molecule ADB-BUTINACA to the positive wells using competitive ELISA to screen the positive hybridoma cells, and select the cells that are sensitive to 400nM ADB-BUTINACA. The cell wells with an inhibition rate of 90% at the ADB-BUTINACA concentration were monocloned by the limiting dilution method until a monoclonal cell line that stably expressed synthetic cannabinoid-specific antibodies was obtained; the cell line was expanded and frozen for use in subsequent experiments.

[0099] Example 10: Large-scale production of antibodies by induction in animals

[0100] Antibodies were prepared in large quantities from 10 6-week-old Balb / c female mice. Prior to hybridoma cell inoculation, each mouse was injected with 0.5 mL of sterile liquid paraffin. One week after liquid paraffin injection, 1 million hybridoma cells with high specificity for ADB-BUTINACA were screened and injected into the peritoneal cavity of the mice to induce peritoneal tumors and ascites. The mice were killed one week later, and the ascites was extracted with a syringe. The supernatant was centrifuged to obtain monoclonal antibodies, which were aliquoted and frozen at -80°C for use after purification.

[0101] Example 11 Protein A purification of ascites antibody (2E4)

[0102] The ascites solution collected in Example 10 was diluted 3 times with a balanced wash solution and loaded onto a Protein A affinity column (Changzhou Tiandi Renhe Biotechnology Co., Ltd.). This was repeated 3 times, and then the impurities were eluted from the affinity column with 5 column volumes of a balanced wash solution (0.15M NaCl, 20mM Na2HPO4, pH 7.0). 2 mL of eluent (0.1M glycine, pH 2.7) was then added each time to elute the antibody from the affinity column. 200 μL of neutralizing solution (1M Tris-HCl, pH 8.5) was added to each collection tube in advance and adjusted to neutrality to obtain an antibody solution. The antibody solution was concentrated using a 50 kD ultrafiltration tube (Millipore) and stored in PBS. The concentration of the antibody solution was measured by NanoDrop to be 9.311 mg / mL, and the solution was aliquoted and frozen for use.

[0103] Example 12 Determination of Antibody Amino Acid Sequence

[0104] The antibody amino acid sequence was determined by hybridoma cell gene sequencing. The monoclonal cell line with high affinity to ADB-BUTINACA obtained by screening was 1.0×10 5The nucleotide sequences of the antibody variable regions were determined (SEQ ID NO. 3 and SEQ ID NO. 4). The present invention provides an amino acid sequence of a synthetic cannabinoid monoclonal antibody for use in constructing a plasmid for rapid, large-scale production of antibodies in a eukaryotic expression system, comprising a 2E4 light chain and a 2E4 heavy chain. The amino acid sequence of the 2E4 light chain variable region is shown in SEQ ID NO.1: ENVLTQSPAIISASPGEKVTMTCSASSSVSYMHWYQQRSSTSPKLWIYDTSKLASGVPGRFSGSGSGNSYSLTISSMEAEDVATYYCFQGSGYPLTFGSGTKLEIK; the amino acid sequence of the 2E4 heavy chain variable region is shown in SEQ ID NO.2: QVQLQQSGPELVKPGASVKMSCKASGYSFTSYYIHWVKQRPGQGLEWIGWIYPGDGSTKYNEKFKGKTTLTADKSSSTAYMLLSSLTSEDSAIYFCVGYYRYDRYHFDYWGQGTTLTVSS.

[0105] The nucleotide sequence encoding the amino acid sequence of the 2E4 light chain variable region as shown in SEQ ID NO.1 is shown in SEQ ID NO.3: GAAAATGTTCTCACCCAGTCTCCAGCAATCATTTCTGCATCTCCAGGGGAAAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAGGTCAAGCACCTCCCCCAAACTCTGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCAGGTCGCTTCAGTGGCAGTGGGTCTGGAAACTCTTACTCTCTCACGATCAGCAGCATGGAGGCTGAAGATGTTGCCACTTATTACTGTTTTCAGGGGAGTGGGTACCCACTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA; the nucleotide sequence encoding the amino acid sequence of the 2E4 heavy chain variable region as shown in SEQ ID NO.2 is shown in SEQ ID Shown in NO.4: CAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACAGCTTCACAAGCTACTATATACACTGGGTGAAACAGCGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAGACGGGAGTACTAAA TACAATGAGAAGTTCAAGGGCAAGACCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGTTGCTCAGCAGCCTGACCTCTGAGGACTCTGCGATCTATTTCTGTGTAGGCTACTATAGGTACGACCGTTATCACTTTGACTATTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

[0106] Example 13 Establishment of a competitive ELISA method based on ADB-BUTINACA monoclonal antibody

[0107] The specific steps of the competitive ELISA method include:

[0108] Coating: Dilute the coating antigen BSA-H08 to 0.25 μg / mL with coating solution, add 100 μl / well to a 96-well ELISA plate, seal with film, and incubate overnight at 4°C. The coating solution contains: 0.2 g KCl, 0.24 g KH2PO4, 3.63 g Na2HPO4.12H2O, 8 g NaCl, and dilute to 1000 mL (pH 7.4) with ddH2O.

[0109] Blocking: Add 100 μl / well of blocking solution and incubate at 37°C for 1.5 h. The blocking solution contains 0.2 g KCl, 0.24 g KH2PO4, 3.63 g Na2HPO4.12H2O, 8 g NaCl, and 50 g skim milk powder. Add ddH2O to 1000 mL (pH 7.4).

[0110] Primary antibody incubation: After blocking, the plate was washed five times with washing buffer, and 50 μl of 12 ng / mL synthetic cannabinoid monoclonal antibody 2E4 and 50 μl of PBS solution containing different concentrations of ADB-BUTINACA (0, 0.098, 0.20, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100 ng / mL) or PBS solution containing different concentrations of ADB-BUTI were added to the wells. NACA test sample solutions (urine and hair samples), where the ADB-BUTINACA concentrations in urine samples were 0, 0.14, 0.41, 1.23, 3.70, 11.11, 33.33, and 100 ng / mL; and the ADB-BUTINACA concentrations in hair samples were 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, and 2.0 ng / mg, incubated at 37°C for 1 h.

[0111] Incubate with secondary antibody: add 100 μL / well of freshly diluted goat anti-mouse HRP enzyme-labeled secondary antibody (PBST: goat anti-mouse HRP enzyme-labeled secondary antibody = 1:5000, v / v) and incubate at 37°C for 30 min;

[0112] Color development: 50 μL / well of TMB color development solution (Biyuntian, P0209 product) was added and incubated in the dark for 10 min;

[0113] Termination: Add 2M H2SO4 (55 mL concentrated H2SO4 plus dd H2O to 500 mL) at 50 μL / well to terminate the color reaction and measure the absorbance at 450 nm.

[0114] Among them, the urine pretreatment step is as follows: artificial urine (Shanghai Yuanye Biotechnology Co., Ltd.) is adjusted to pH 7.4 using 1M NaOH solution, and added with or without ADB-BUTINACA to a 96-well ELISA plate containing the synthetic cannabinoid monoclonal antibody 2E4 for incubation and detection.

[0115] Hair pretreatment: Weigh 20 mg of clean, chopped blank hair (provided by laboratory staff) and add it to a grinding tube containing 0.8 mL of hair lysis buffer and grinding beads, with or without ADB-BUTINACA. Grind the hair in a hair grinder for 10 minutes, centrifuge, and add the supernatant to a 96-well ELISA plate containing 2E4 for incubation.

[0116] The results of PBS-diluted samples (synthetic cannabinoid monoclonal antibody 2E4 and PBS solutions containing different concentrations of ADB-BUTINACA were added to the wells) are shown in Figure 13. In PBS solution, the IC values ​​of synthetic cannabinoid monoclonal antibody 2E4 and ADB-BUTINACA were 50 The value is 2.295ng / mL.

[0117] The results of competitive ELISAs for urine and hair samples (with synthetic cannabinoid monoclonal antibody 2E4 and test sample solutions containing varying concentrations of ADB-BUTINACA added to the wells, respectively) are shown in Figure 14. Figures A and B show the results of competitive ELISAs for hair and urine, respectively, with concentration on the abscissa and OD450 values ​​on the ordinate. Using the competitive ELISA method, the LOD for hair detection was 0.02 ng / mg, and for urine detection was 0.389 ng / mL.

[0118] Example 14 Establishment of Colloidal Gold Immunochromatography Method Based on ADB-BUTINACA Monoclonal Antibody

[0119] Step 1: 0.8 mg / mL BSA-H08 coating antigen and 0.2 mg / mL goat anti-mouse secondary antibody (Jackson ImmunoResearch, 205-035-108) were streaked onto the NC membrane using a gold streaker at 1 μL / cm and dried at 37°C. These served as the test line T and the quality control line C, respectively.

[0120] Step 2: Assemble the sample pad, NC membrane, and absorbent pad on the PVC base plate in order, and cut them into 3mm wide test strips using a chopper;

[0121] Step 3: 50 μL of urine and hair sample processed solutions containing different concentrations of ADB-BUTINACA and 50 μL of colloidal gold solution modified with antibody 2E4 (3 μL of gold-labeled antibody stock solution mixed with 47 μL of PBS) were mixed and incubated in a 96-well plate for 2 minutes. The test strip was inserted into the microwell, and the mixed solution moved along the chromatography direction under the action of capillary siphon. When it reached the test line T and the quality control line C, the ratio of the test line and the quality control line was read by the colloidal gold reader TSR-100 (Hangzhou Aosheng Instrument Co., Ltd.). According to the different ADB-BUTINACA concentrations, the different values ​​read by the test line T and the quality control line C were obtained, and the ratio T / C of the test line and the quality control line was obtained. The standard curve was made by using this ratio and the corresponding ADB-BUTINACA concentration to achieve the detection of this synthetic cannabinoid. Among them, the ADB-BUTINACA concentrations in urine samples were 0, 2, 5, 10, 20, 50, 100, 200, 500, and 1000 ng / mL; the ADB-BUTINACA concentrations in hair samples were 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, and 50 ng / mg.

[0122] Preparation of colloidal gold solution modified with antibody 2E4: Rapidly add 2 mL of 1% trisodium citrate aqueous solution to 100 mL of boiling 0.01% HAuCl₄ aqueous solution and continue boiling with stirring for 15 minutes. The colloidal gold solution is then cooled to room temperature and stored at 4°C. Antibody 2E4 is attached to the colloidal gold using electrostatic adsorption. First, pipette 1 mL of colloidal gold solution into a 1.5 mL EP tube. Adjust the pH of the colloidal gold solution to 8.2 with 0.1 mol / L K₂CO₃. Then, add 8 μg of synthetic cannabinoid antibody 2E4 and incubate at room temperature for 1 hour under rotating rotation. Then, add bovine serum albumin (BSA) dissolved in 0.01 M boric acid buffer (BB, pH 8.2) to a final concentration of 10 mg / mL and incubate for an additional hour. After centrifugation at 10,000 rpm / min for 15 minutes, the precipitate was resuspended in 100 μL BB solution containing 2% bovine serum albumin and 3% sucrose to obtain the gold-labeled antibody stock solution.

[0123] The pretreatment steps for the colloidal gold immunochromatographic assay for urine and hair samples were the same as those in Example 13. The results are shown in Figure 15 , where A, C, and E are colloidal gold test strip images of PBS, urine, and hair samples, respectively, and B, D, and F are numerical graphs of the colloidal gold test strip images of PBS, urine, and hair samples, respectively. The abscissa represents concentration, and the ordinate represents T / C. As the ADB-BUTINACA concentration increases, the T / C value gradually decreases, indicating that the established colloidal gold test strip immunochromatographic assay is capable of quantifying ADB-BUTINACA in urine and hair.

[0124] Example 15 HEK 293F cells expressing ADB-BUTINACA monoclonal antibody

[0125] 1. Plasmid construction

[0126] The full-length expression plasmids of the light and heavy chains of the ADB-BUTINACA-specific monoclonal antibody 2E4 were constructed separately: the expression vector was pcDNA3.4, the constant region sequences were mouse IgG1 and kappa (from Nanjing Detai Biotechnology Co., Ltd.), the signal peptide sequence was selected as MGWSCIILFLVATATGVHS (SEQ ID NO.5), the connection method was shown in Figure 16, and gene synthesis was completed by Detai Biotechnology (Nanjing) Co., Ltd. The signal peptide + light chain variable region or heavy chain variable region + mouse constant region (IgG1 constant region and kappa constant region) was connected to the pcDNA3.4 vector to obtain light and heavy chain plasmids. Among them, the nucleotide sequence of the signal peptide is shown in SEQ ID NO.6: ATGGGCTGGAGCTGCATCATCCTGTTCCTCGTGGCTACAGCTACCGGAGTGCACAGC. The constant region sequence mouse IgG1 sequence is shown as follows: SEQ IDNO.7: GCTAAGACAACCCCTCCTTCTGTGTACCCTCTGGCTCCAGGAAGCGCAGCTCAGACCAACAGCATGGTGACACTGGGCTGCCTGGTGAAGGGATACTTCCCCGAGCCAGTGACCGTGACTTGGAACTCAGGCAGCCTGAGCAGCGGAGTGCACACATTTCCAGCCGTGCTGCAGAGCGACCTGTACACACTGAGCAGCAGCGTGACCGTGCCTTCTAGCACTTGGCCTAGCGAGACCGTGACTTGCAACGTGGCTCACCCAGCCAGCAGCACCAAGGTGGACAAGAAGATCGTGCCCAGGGATTGCGGTTGCAAGCCTTGCATCTGTACCGTGCCCGAAGTGTCCAGCGTGTTCATCTTCCCCCCCAAGCCCAAGGACGTGCTGACCATCACCCTGACCCCCAAAGTGACTTGCGTGGTGGTGGACATCAGCAAGGACGACCCCGAGGTGCAGTTCTCTTGGTTCGTGGACGACGTGGAGGTGCACACAGCTCAGACACAGCCTAGGGAGGAGCAGTTCAACAGCACCTTCCGGAGCGTGTCCGAACTGCCCATCATGCACCAGGATTGGCTGAACGGCAAGGAGTTCAAGTGTCGCGTGAACAGCGCCGCTTTCCCAGCCCCCATCGAGAAGACCATCAGCAAGACCAAGGGCAGGCCTAAGGCTCCTCAGGTGTACACAATCCCTCCTCCCAAGGAGCAGATGGCCAAGGACAAGGTGTCCCTGACTTGCATGATCACCGACTTCTTCCCCGAGGACATCACCGTCGAGTGGCAGTGGAACGGACAGCCAGCCGAGAACTACAAGAACACCCAGCCCATCATGGACACCGACGGCAGCTACTTCGTGTACAGCAAACTGAACGTGCAGAAGAGCAATTGGGAGGCCGGCAACACCTTCACTTGCAGCGTGCTGCACGAGGGACTGCACAACCACCACACCGAGAAGAGCCTGAGCCACAGCCCAGGAAAG. The constant region mouse kappa sequence is as follows: SEQID NO.8: AGGGCCGACGCAGCTCCTACCGTGTCTATCTTCCCCCCTAGCAGCGAGCAGCTGACATCAGGAGGAGCTAGCGTGGTCTGCTTCCTGAACAACTTCTACCCCAAGGACATCAACGTCA AGTGGAAGATCGACGGCAGCGAGAGGCAGAACGGCGTGCTGAACTCTTGGACCGACCAGGATAGCAAGGACAGCACCTACAGCATGAGCAGCACCCTGACCCTGACCAAGGACGAGTACGAGCG GCACAACAGCTACACCTGCGAGGCTACACACAAGACCAGCACCAGCCCCATCGTGAAGAGCTTCAACCGGAACGAGTGC.

[0127] 2. Plasmid transformation

[0128] Prepare the required solid culture medium in advance. Weigh 1g sodium chloride, 1g peptone, 0.5g yeast extract, and 1.5g agar powder. Add 100mL of ultrapure water. After sterilization, cool to 50°C. Then, add 100μL of ampicillin solution (100mg / mL). Mix thoroughly, pour into a Petri dish, and wait for solidification. Seal the container and refrigerate at 4°C until ready to use.

[0129] The received 2E4 light and heavy chain full-length expression plasmids were diluted with sterile water to 1 ng / mL plasmid solution; 50 μL competent cells DH5α (Shanghai Weidi Biotechnology Co., Ltd.) were taken out from the -80°C refrigerator and sterilized to 1.5 mL EP tube, add 1 μL of plasmid solution, stir gently with a pipette tip to mix, and let it stand on ice for 30 minutes; after 30 minutes, transfer it to a 42℃ water bath for heat shock for 90 seconds and then quickly put it back on ice and let it stand for 2 minutes; after 2 minutes, add 200 μL of sterilized LB medium, put it in a shaker, shake at 37℃, 180rpm for 60 minutes; spread the bacterial solution on the prepared solid culture plate, invert and culture overnight at 37℃; pick up the grown monoclonal colony with a pipette tip, add it to LB medium containing 1% ampicillin, put it in a shaker at 37℃, 180rpm for 5 hours, take the bacterial solution that can be successfully amplified and send it to Beijing Qingke Biotechnology Co., Ltd. for sequencing to verify whether the transformation is correct, mix the bacterial solution with sterilized 50% glycerol in a 1:1 ratio, and freeze it in a -80℃ refrigerator.

[0130] 3. Plasmid Extraction

[0131] Sequence-verified full-length expression plasmid solutions of the 2E4 light and heavy chains were removed from a −80°C freezer, placed in LB medium containing 1% ampicillin (Shanghai Beyotime Biotechnology Co., Ltd.), and shaken at 180 rpm at 37°C for 14 h. The culture was removed the next day and operated using a plasmid extraction kit (Kangwei Century Biotechnology Co., Ltd.). After measuring the plasmid concentration using a NanoDrop assay, the extracted full-length expression plasmid concentration of the 2E4 light chain was 720.751 ng / μL, and the heavy chain plasmid concentration was 614.737 ng / μL. The cells were then frozen in a −20°C freezer until use.

[0132] 4. Cell culture

[0133] Remove the frozen HEK 293F cells from the -80°C freezer and quickly place them in a preheated 37°C water bath. Shake back and forth continuously until there are no ice crystals in the cryotube. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL of OPM-293CD05 Medium to resuspend the cells, and transfer them to a shake flask containing OPM-293CD05 Medium (Shanghai Aopmin Biotech Co., Ltd.). Shake at 120 rpm, set the temperature to 37°C, and culture overnight in 5% CO2.

[0134] 5. Cell transfection

[0135] When the density of HEK 293F cells in 300 mL reaches 3×10 6 cells / mL, start transfection. Add 150 μg of each of the full-length expression plasmids of the 2E4 light and heavy chains to 10 mL of blank culture medium, and slowly drop 1.2 mg of 1

[0136] 100 mg / mL PEI transfection reagent (PolySciences) was vortexed and allowed to stand for 15 minutes before being slowly added to the cell culture medium. Five days after transfection, the cell supernatant was collected and purified.

[0137] 6. Antibody Purification

[0138] The cell supernatant was collected by centrifugation at 8000 rpm at 4°C, filtered through a 0.22 μM filter membrane, and purified using Protein A. The subsequent steps were the same as in Example 11.

[0139] 7. Validation of eukaryotic expression antibodies

[0140] The operation steps are the same as those in Example 13. The specific competitive concentrations of ADB-BUTINACA are 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, and 0 ng / mL. The results are shown in FIG17 . The IC values ​​of the purified 2E4 antibody expressed in the eukaryotic system and ADB-BUTINACA are shown in FIG17 .50 The value was 5.692 ng / mL, and the results showed that the affinity of the antibody prepared by hybridoma cell ascites was similar to that of ADB-BUTINACA, indicating that the plasmid can correctly express the specific antibody in the eukaryotic system. Example 16 2E4 Antibody Specificity Detection

[0141] Specificity analysis was performed using a competitive ELISA method with the same operating steps as in Example 13. The competitive concentrations of synthetic cannabinoids were 200, 66.67, 22.22, 7.41, 2.47, 0.82, 0.27, and 0 ng / mL, respectively, and the IC 50 IC values ​​of mAb 2E4 and various synthetic cannabinoids 50 The values ​​are shown in Table 1. All synthetic cannabinoids used were obtained from the Joint Laboratory of Key Drug Control Technologies of the Office of the National Narcotics Control Commission and China Pharmaceutical University.

[0142] Table 1 2E4 and synthetic cannabinoid IC 50 value

[0143] The results indicate that the monoclonal antibody 2E4 can recognize multiple synthetic cannabinoids and their metabolites, and can be used to establish a rapid detection method for the simultaneous detection of multiple synthetic cannabinoids and their metabolites.

Claims

1. A synthetic cannabinoid hapten compound, characterized in that Its general structural formula is shown below: Among them, Linker is a single bond, double bond, triple bond, C 1-8 Hydrocarbon, with or without amino groups 1-8 Hydrocarbon groups, with or without amide bonds 1-8 a hydrocarbon group, p-phenyl, m-phenyl, o-phenyl, 1,4-divinylphenyl, vinylphenyl, ethynylphenyl, 1,4-diethynylphenyl, biphenyl, 1,4-methylenephenyl, or 9,10-anthryl.

2. A synthetic cannabinoid hapten compound, characterized in that Its structure is as follows:

3. Use of the synthetic cannabinoid hapten according to any one of claims 1 to 2 in the preparation of a synthetic cannabinoid complete antigen.

4. A synthetic cannabinoid complete antigen prepared from the synthetic cannabinoid hapten compound according to any one of claims 1 to 2, characterized in that: Its structure is as follows:

5. Use of the synthetic cannabinoid hapten according to any one of claims 1 to 2 or the synthetic cannabinoid complete antigen according to claim 4 in the preparation of a synthetic cannabinoid monoclonal antibody.

6. A synthetic cannabinoid monoclonal antibody, characterized in that The synthetic cannabinoid complete antigen is prepared by animal immunization.

7. The synthetic cannabinoid monoclonal antibody according to claim 6, characterized in that The amino acid sequence of the light chain variable region is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.3; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.

4.

8. A colloidal gold immunochromatographic test strip, characterized in that: It contains the synthetic cannabinoid monoclonal antibody according to claim 6.

9. Use of the synthetic cannabinoid hapten compound according to any one of claims 1 to 2, the synthetic cannabinoid complete antigen according to claim 4, the synthetic cannabinoid monoclonal antibody according to any one of claims 6 to 7, or the colloidal gold immunochromatographic test strip according to claim 8 in detecting the content of synthetic cannabinoids.

10. The use according to claim 9, characterized in that The synthetic cannabinoids include one or more of ADB-BUTINACA, ADB-CHMINACA, ADB-CHMICA, AB-FUBINACA, AB-CHMINACA, ADB-3en-BUTINACA, AB-4en-PINACA, ADB-4en-PINACA, EMB-FUBINACA or MDMB-BUTINACA butanoic acid metabolites.

11. A plasmid vector, characterized in that: It contains the nucleotide sequence shown in SEQ ID NO. 3 or the nucleotide sequence shown in SEQ ID NO. 4 according to claim 7.

12. A method for constructing the plasmid vector according to claim 11, characterized in that: The method comprises the following steps: using pcDNA3.4 as an expression vector, the nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8 as constant region sequences, the nucleotide sequence shown in SEQ ID NO.6 as a signal peptide sequence, and the nucleotide sequence shown in SEQ ID NO.3 or the nucleotide sequence shown in SEQ ID NO.4 as the variable region sequence as claimed in claim 11.

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