Hapten for synthetic cannabinoids as well as monoclonal antibody thereof and use thereof

Abstract

Provided are a hapten for synthetic cannabinoids, a monoclonal antibody thereof, and the use thereof, the structural general formula of the hapten for synthetic cannabinoids being as shown in formula (I). The present invention successfully obtains an antibody specifically targeting one or more synthetic cannabinoids such as 4F-MDMB-BUTICA, and the antibody obtained after purification is successfully applied to development of an enzyme-linked immunosorbent assay and a colloidal gold immunochromatographic assay for measuring the content of one or more synthetic cannabinoids such as 4F-MDMB-BUTICA. The present invention, for the first time, obtains the amino acid sequence of a 4F-MDMB-BUTICA specific monoclonal antibody; and the specific monoclonal antibody is successfully expressed via a eukaryotic system.

Description

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

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

[0002] Synthetic cannabinoids have the hallucinogenic effects of plant cannabis. They are a class of substances that act by simulating the effects of tetrahydrocannabinol (THC), the main psychoactive ingredient in cannabis. They are also agonists of the cannabinoid receptors CB1 and CB2 in the body. Synthetic cannabinoids can affect the function of the central nervous system. Long-term use can lead to low immunity, induce mental confusion and suicidal tendencies, and excessive use can cause shock, suffocation, and even sudden death. It can also cause incidents that endanger public safety, such as drug driving or intentional injury. Data show that in recent years, new synthetic cannabinoid drugs have spread rapidly in my country, and the amount of such substances detected has accounted for more than 50% of all new psychoactive substances detected in the country. Starting from July 1, 2021, the country will list synthetic cannabinoids as new psychoactive substances as a whole category, making my country the first country in the world to list synthetic cannabinoids as a whole category.

[0003] Patent applications (application numbers: 2021112959476, 2021112942117, 2021112943035, 2022113439384) disclose synthetic cannabinoid hapten compounds, but these compounds do not involve the detection of the three most recently abused synthetic cannabinoids, such as 5F-MDMB-PICA, 5F-EMB-PICA, and 4F-MDMB-BUTICA.

[0004] Summary of the Invention

[0005] 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.

[0006] Technical solution: To solve the above technical problems, the present invention provides a synthetic cannabinoid hapten compound, the structure of which is shown below:

[0007] Among them, Linker is a single bond, double bond, triple bond, C 1-8 Hydrocarbon, C with or without amino 1-8 The hydrocarbon group, p-phenyl, m-phenyl, o-phenyl;

[0008] R1 is substituted or unsubstituted C6-C 10Aryl, substituted or unsubstituted C3-C 10 of hydrocarbon groups;

[0009] R2 is a single bond, a double bond, a triple bond, a heteroatom / halogen substituted or unsubstituted alkyl group, a substituted or unsubstituted heterocyclic group containing 1 to 3 heteroatoms / halogens, a methyl group or an ethyl group.

[0010] The present invention also provides the use of the synthetic cannabinoid hapten in the preparation of a synthetic cannabinoid complete antigen.

[0011] The present invention also provides a synthetic cannabinoid complete antigen obtained from the synthetic cannabinoid hapten compound, the structure of which is shown below:

[0012] Among them, Linker is a single bond, double bond, triple bond, C 1-8 Hydrocarbon, with or without amino groups 1-8 The hydrocarbon group, p-phenyl, m-phenyl, o-phenyl;

[0013] R1 is substituted or unsubstituted C6-C 10 Aryl, substituted or unsubstituted C3-C 10 of hydrocarbon groups;

[0014] R2 is a single bond, a double bond, a triple bond, a heteroatom / halogen substituted or unsubstituted alkyl group, a substituted or unsubstituted heterocyclic group containing 1 to 3 heteroatoms / halogens, a methyl group or an ethyl group.

[0015] The present invention also provides a synthetic cannabinoid monoclonal antibody, which is prepared by immunizing animals with the synthetic cannabinoid complete antigen.

[0016] Among them, 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.

[0017] The present invention also provides the use of the synthetic cannabinoid hapten or the synthetic cannabinoid complete antigen in the preparation of synthetic cannabinoid monoclonal antibodies.

[0018] The present invention also provides the use of the synthetic cannabinoid hapten compound or the synthetic cannabinoid complete antigen or the synthetic cannabinoid monoclonal antibody in detecting the content of synthetic cannabinoids.

[0019] The present invention also provides a colloidal gold immunochromatographic test strip containing the synthetic cannabinoid monoclonal antibody.

[0020] The present invention also provides the use of the colloidal gold test strip in detecting the content of synthetic cannabinoids.

[0021] Wherein, the synthetic cannabinoids include 4F-MDMB-BUTICA, ADB-BUTINACA, ADB-CHMICA, AB-FUBINACA, AB-CHMINACA, ADB-3en-BUTINACA, AB-4en-PINACA, ADB-4en-PINACA, ADB-P7AICA, 5F-AMB-PICA, 5F- EMB-PICA, 5F-MDMB-PICA, 5F-MPP-PICA, 5F-APP-PICA, 5F-EDMB-PICA, 5Br-AMB-PICA, 4Cl-MDMB-PICA, AMB-4en-PICA, AMB-CHMICA, MDMB-4en-PICA, MPP-PICA, MDMB-4en-PINACA butanoic acid metabolite, MDMB-BUTINACA butanoic acid metabolite, 5-fluoro MDMB-PICA metabolite 7, 4F-MDMB-BUTICA butanoic acid One or more of metabolite or 4-fluoro MDMB-BINACA 3,3-dimethylbutanoic acid.

[0022] The present invention also provides a plasmid vector containing the nucleotide sequence shown in SEQ ID NO.3 or the nucleotide sequence shown in SEQ ID NO.4.

[0023] 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, 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 described in claim 12.

[0024] The present invention designs haptens and prepares antibodies based on the structures of three target synthetic cannabinoids, 5F-MDMB-PICA, 5F-EMB-PICA, and 4F-MDMB-BUTICA, and then uses the prepared antibodies to establish a detection method to provide assistance for the monitoring of synthetic cannabinoids.

[0025] Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages: 1. The present invention successfully obtains antibodies specific to one or more synthetic cannabinoids such as 4F-MDMB-BUTICA, and successfully applies the purified antibodies to the development of enzyme-linked immunosorbent assay and colloidal gold immunochromatography for detecting the content of one or more synthetic cannabinoids in 4F-MDMB-BUTICA; 2. The present invention also reports the amino acid sequence of the 4F-MDMB-BUTICA-specific monoclonal antibody for the first time; 3. The specific monoclonal antibody is successfully expressed in a eukaryotic system. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 shows the hapten of the synthetic cannabinoid compound H-5F-1. 1 H NMR spectrum;

[0027] Figure 2 shows the hapten of the synthetic cannabinoid compound H-5F-1. 13 C NMR spectrum;

[0028] FIG3 is a high-resolution mass spectrum of the synthetic cannabinoid hapten compound H-5F-1;

[0029] Figure 4 shows the hapten of synthetic cannabinoid compound H-5F-2. 1 H NMR spectrum;

[0030] Figure 5 shows the hapten of synthetic cannabinoid compound H-5F-2. 13 C NMR spectrum;

[0031] FIG6 is a high-resolution mass spectrum of the synthetic cannabinoid hapten compound H-5F-2;

[0032] Figure 7 shows the synthetic cannabinoid hapten compound H-4F 1 H NMR spectrum;

[0033] Figure 8 shows the hapten of synthetic cannabinoid compound H-4F. 13 C NMR spectrum;

[0034] FIG9 is a high-resolution mass spectrum of the synthetic cannabinoid hapten compound H-4F;

[0035] Figure 10 shows the antibody titers of mice immunized 21 and 35 days after the complete synthetic cannabinoid antigen was mixed with an adjuvant;

[0036] Figure 11 shows the affinity of antibodies to 5F-MDMB-PICA, 5F-EMB-PICA, and 4F-MDMB-BUTICA 35 days after immunization of mice with complete synthetic cannabinoid antigens mixed with adjuvants;

[0037] FIG12 shows the competitive ELISA results of the purified monoclonal antibody mAb-4F and synthetic cannabinoids for IC50 value;

[0038] FIG13 shows the affinity of the purified monoclonal antibody mAb-4F to the synthetic cannabinoid 4F-MDMB-BUTICA measured by ITC: A is the ITC titration graph; B is the fitting curve of the titration results;

[0039] FIG14 is a standard curve of 4F-MDMB-BUTICA in hair and urine determined by ELISA;

[0040] FIG15 is a colloidal gold immunochromatographic assay for the determination of 4F-MDMB-BUTICA in hair and urine: A, C, and E are colloidal gold test strips for the PBS, urine, and hair standard curves, respectively; B, D, and F are colloidal gold test strips for the PBS, urine, and hair standard curves, respectively, and T / C curves;

[0041] Figure 16 is a schematic diagram of the construction of light and heavy chain expression vectors;

[0042] FIG17 is a validation of the activity of synthetic cannabinoid antibodies expressed in HEK 293F cells. DETAILED DESCRIPTION

[0043] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0044] Example 1 Preparation of synthetic cannabinoid hapten H-5F-1

[0045] Synthesis route:

[0046] 1. Condensation reaction:

[0047] Indole-3-carboxylic acid (500 mg, 1 eq), carbodiimide hydrochloride (EDCI) (892 mg, 1.5 eq), and 1-hydroxybenzotriazole (HOBt) (629 mg, 1.5 eq) were mixed and dissolved in 5 mL of N,N-dimethylformamide (DMF). The mixture was stirred under N2 protection and the reaction mixture was stirred for half an hour. L-tert-leucine methyl ester hydrochloride (677 mg, 1.5 eq) was weighed and dissolved in 3 mL of DMF. The mixture was added to the reaction mixture along with N,N-diisopropylethylamine (DIPEA) (1204 mg, 3 eq) and stirred at room temperature for 4 h. After TLC confirmed that the reaction of the starting materials was complete, the reaction was terminated with ethyl acetate. The mixture was washed twice 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 = 4:1, v / v) to obtain intermediate 1.

[0048] 2. Substitution reaction:

[0049] Weigh NaH (85 mg, 2 eq) and dissolve it in DMF, add intermediate 1 (500 mg, 1 eq), and stir the reaction solution for 0.5 h under N2 protection and ice bath conditions; weigh 1-bromo-5-fluoropentane (381 mg, 1.2 eq) and dissolve it in 3 mL DMF, add it to the reaction solution, continue stirring at room temperature until the reaction is complete, terminate the reaction with ethyl acetate, wash twice with saturated sodium bicarbonate and once with saturated sodium chloride, take the ethyl acetate layer, dry it over anhydrous sodium sulfate, concentrate it by rotary evaporation, add silica gel powder and spin dry it, and pass it through a silica gel chromatography column (developing solvent petroleum ether: ethyl acetate = 10:1, v / v) to obtain intermediate 2.

[0050] 3. Ester hydrolysis reaction:

[0051] Intermediate 2 (500 mg, 1 eq) was dissolved in 10 mL of methanol and 100 μL of saturated KOH was added. The reaction was stirred at room temperature and monitored by TLC until the reaction was complete. The remaining methanol was removed by rotary evaporation, and an appropriate amount of pure water was added to dissolve the solid. While stirring in 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 dried to obtain Intermediate 3.

[0052] 4. Condensation reaction and ester hydrolysis reaction:

[0053] Intermediate 3 (250 mg, 1 eq), EDCI (198 mg, 1.5 eq), and HOBt (140 mg, 1.5 eq) were mixed and dissolved in 5 mL of DMF. The mixture was stirred under N2 protection for half an hour. 6-Aminohexanoic acid methyl ester hydrochloride (150 mg, 1.5 eq) was weighed and dissolved in 3 mL of DMF. This was added to the reaction solution along with 0.36 mL of DIPEA and stirred at room temperature for 4 hours. TLC confirmed complete reaction of the starting materials and terminated the reaction with ethyl acetate. The mixture was washed twice with saturated sodium bicarbonate and once with saturated sodium chloride. The ethyl acetate layer was dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and then dried over silica gel powder. The mixture was then passed through a silica gel chromatography column (developing solvent: petroleum ether:ethyl acetate = 4:1, v / v). The resulting intermediate was subjected to ester hydrolysis to yield the final product, H-5F-1. The corresponding H-1R, C-1R, and high-resolution mass spectra are shown in Figures 1 to 3, respectively.

[0054] 1H NMR(300MHz, DMSO-d6)δ12.02(s,1H),8.34(s,1H),8.21–7.95(m,2H),7.56(d,J=7.8Hz,1H),7.36–7.02(m,3H),4.57–4.44(m,2H),4.34(t, J=6.0Hz,1H),4.23(t,J=6.3Hz,2H),3.07(m,2H),2.17(t,J=7.3Hz,2H),1.85(p,J=7.3Hz,2H),1.67(m,2H),1.55–1.20(m,8H),0.99(s,9H).

[0055] 13 C NMR(75MHz,DMSO-d6)δ174.90,170.66,164.24,136.59,132.08,122.39,121.26,121.16,110.95,109.71, 85.20,83.05,59.74,46.21,38.73,34.85,34.06,29.98,29.73,29.22,27.36,26.49,24.63,22.64,22.56.

[0056] HRMS(ESI)m / z:[M+H] + calculated=476.2925; found=476.2920.

[0057] Example 2 Preparation of Synthetic Cannabinoid Hapten H-5F-2

[0058] Synthesis route:

[0059] 1. Condensation reaction:

[0060] The L-tert-leucine methyl ester hydrochloride in Example 1 was replaced with L-valine ethyl ester hydrochloride of equimolar mass to obtain intermediate 4.

[0061] 2. Substitution reaction:

[0062] Intermediate 5 was obtained by replacing Intermediate 1 in Example 1 with Intermediate 4 of equal molar mass and using the same raw materials and steps as Step 2 in Example 1.

[0063] 3. Ester hydrolysis reaction:

[0064] Intermediate 6 was obtained by replacing Intermediate 2 in Example 1 with Intermediate 5 of equal molar mass and using the same raw materials and steps as Step 3 in Example 1.

[0065] 4. Condensation reaction and ester hydrolysis reaction:

[0066] The intermediate 3 in Example 1 was replaced with an intermediate 6 of equal molar mass. The other raw materials and steps were the same as those in Step 4 of Example 1 to obtain the final product H-5F-2. The corresponding H-1N spectrum, C-1N spectrum, and high-resolution mass spectrum are shown in Figures 4 to 6, respectively.

[0067] 1 H NMR (300MHz, DMSO-d6) δ12.03(s,1H),8.28(s,1H),8.19–7.97(m,2H),7.58(m,2H),7.17(m,2H),4.50(t,J=5.9Hz,1H),4 .38–4.14(m,4H),3.08(m,2H),2.11(dt,J=37.5,6.9Hz,3H),1.91–1.56(m,4H),1.55–1.14(m,8H),0.93(d,J=4.4Hz,6H).

[0068] 13 C NMR(75MHz,DMSO-d6)δ174.93,171.80,164.48,136.54,131.76,127.01,122.36,121.54,121.07,110.83,109.78, 85.20,83.05,58.29,46.21,38.74,34.08,30.97,29.98,29.72,29.24,26.41,24.65,22.65,22.58,19.89,19.25.

[0069] HRMS(ESI)m / z:[M+H] + calculated=462.2768; found=462.2837.

[0070] Example 3 Preparation of synthetic cannabinoid hapten H-4F

[0071] Synthesis route:

[0072] 1. Condensation reaction:

[0073] Same as Example 1.

[0074] 2. Substitution reaction:

[0075] The 1-bromo-5-fluoropentane in Example 1 was replaced with 1-bromo-4-fluorobutane of equal molar mass. Other raw materials and steps were the same as those in Step 2 of Example 1 to obtain Intermediate 7.

[0076] 3. Ester hydrolysis reaction:

[0077] Intermediate 8 was obtained by replacing Intermediate 2 in Example 1 with Intermediate 7 of equal molar mass and using the same raw materials and steps as Step 3 in Example 1.

[0078] 4. Condensation reaction and ester hydrolysis reaction:

[0079] The intermediate 3 in Example 1 was replaced with the intermediate 8 of equal molar mass. The other raw materials and steps were the same as those in Step 4 of Example 1 to obtain the final product H-4F. The corresponding H-1N spectrum, C-1N spectrum, and high-resolution mass spectrum are shown in Figures 7 to 9, respectively.

[0080] 1 H NMR(300MHz,DMSO-d6)δ11.96(s,1H),8.35(s,1H),8.19–8.03(m,2H),7.57(d,J=8.0Hz ,1H),7.33–7.10(m,3H),4.60–4.46(m,2H),4.39(t,J=6.0Hz,1H),4.26(t,J=7.2Hz,2H) ,3.07(ddq,J=46.7,12.4,6.6Hz,2H),2.17(t,J=7.3Hz,2H),1.90(p,J=7.0Hz,2H),1.7 5–1.56(m,2H),1.45(dp,J=22.4,7.0Hz,4H),1.28(dd,J=15.6,8.4Hz,3H),0.99(s,9H). 13 C NMR (75MHz, DMSO-d6) δ175.07,170.67,164.25,136.56,132.07,126.82,122.43,121.30,121.20,110.92,109. 79,84.99,82.84,59.81,45.90,38.72,34.85,34.36,29.19,27.82,27.56,27.36,26.52,26.14,26.07,24.70.

[0081] HRMS(ESI)m / z:[M+H] + calculated=462.2768; found=462.2705.

[0082] Example 4 Synthesis of three synthetic cannabinoid complete antigens

[0083] 5 mg each of H-5F-1, H-5F-2 and H-4F were weighed, and EDCI (6 eq), NHS (6 eq) and Et3N (6 eq) were dissolved in 500 μL DMF with H-5F-1, H-5F-2 and H-4F, respectively. The mixture was stirred at room temperature for 6 h until the hapten reaction was complete. 5 mg / mL BSA solution (10 mg BSA was dissolved in 2 mL 0.01 M, pH 7.4 PBS solution) or KLH solution (10 mg KLH was dissolved in 2 mL 0.01 M, pH 7.4 PBS solution) was added, respectively. The mixture was stirred at 4 ° C overnight. The mixture was taken out the next day and placed in a MW 3000 dialysis bag. 500 mL pH 7.4 was used each time. The solution was dialyzed against 7.4% PBS four times to obtain the immune antigens KLH-H-5F-1, KLH-H-5F-2, and KLH-H-4F, and the coating antigens BSA-H-5F-1, BSA-H-5F-2, and BSA-H-4F. Protein concentrations were determined by BCA assay: KLH-H-5F-1, KLH-H-5F-2, and KLH-H-4F concentrations were 1.33, 1.53, and 1.22 mg / mL, respectively; and BSA-H-5F-1, BSA-H-5F-2, and BSA-H-4F concentrations were 0.85, 0.97, and 1.18 mg / mL, respectively. Aliquots were frozen at -80°C until use. Among them, the immune antigens KLH-H-5F-1, KLH-H-5F-2, KLH-H-4F and the coating antigens BSA-H-5F-1, BSA-H-5F-2, BSA-H-4F are all complete antigens. The structural formula of the complete antigen is as follows:

[0084] Wherein, KLH / BSA in the above structural formula represents KLH protein or BSA protein.

[0085] Example 5 Immunization with three complete antigens

[0086] Six-week-old Balb / c female mice were subcutaneously injected with multiple points on the back of the mice. The three immune antigens, KLH-H-5F-1, KLH-H-5F-2, and KLH-H-4F, were injected at a dose of 0.1 mL / 50 μg / mouse. During the first immunization, 100 μg of the three synthetic cannabinoid immune antigens, KLH-H-5F-1, KLH-H-5F-2, and KLH-H-4F, were emulsified in equal volumes with complete Freund's adjuvant (Sigma, F5581). Two weeks later, a second booster immunization was performed. 50 μg of the three synthetic cannabinoid immune antigens, KLH-H-5F-1, KLH-H-5F-2, and KLH-H-4F, were emulsified in equal volumes with incomplete Freund's 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 mouse orbits on the 21st and 35th day after the initial immunization, and the serum was collected and aliquoted and frozen at -80°C for use.

[0087] Example 6 Determination of mouse serum antibody titer

[0088] 1. Serum titer determination:

[0089] Coating: Dilute the coating antigens BSA-H-5F-1, BSA-H-5F-2, and BSA-H-4F to 1 μg / ml in 0.01 M, pH 7.4 PBS solution, add 100 μl / well to a 96-well ELISA plate, seal the plate with film, and incubate in a 4°C refrigerator overnight.

[0090] Blocking: Wash the plate three times with PBST, add 100 μl / well of blocking solution, and incubate at 37°C for 1.5 h.

[0091] Primary antibody incubation: After blocking, wash the plate five times with PBST, and add 100 μl of serially diluted serum (starting from 1:1000 with PBST, diluted 3-fold, 8 concentrations are 1:1000, 1:3000, 1:9000, 1:27000, 1:81000, 1:243000, 1:729000, 1:2187000) and blank control serum to each well;

[0092] 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.

[0093] 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;

[0094] Termination: Add 2M H2SO4 to terminate the color reaction (50μL / well) and use an MD i3x microplate reader to measure the absorbance at a wavelength of 450nm. The OD450 value at the maximum serum dilution is 2.1 times higher than that of the blank serum, which is the antibody titer. The results are shown in Figure 10. Comparing the antibody titers at 21 days and 35 days, after immunization of mice with the three antigens, the hapten H-4F can produce the highest antibody titer, reaching 10 at 35 days. 6 , while the antibody titers produced by H-5F-1 and H-5F-2 were both above 10 5 .

[0095] 2. Affinity determination with synthetic cannabinoids

[0096] The implementation steps are the same as those for antibody titer determination, except that when incubating with the primary antibody, the steps are replaced by "adding 50 μL of serum of a certain dilution (dilution at an OD value of 1) and 50 μL of different concentrations of synthetic cannabinoids (laboratory-preserved) for co-incubation. The competing concentrations of synthetic cannabinoids are 400, 100, 25, 6.25, 1.56, 0.391, 0.098, and 0 μM, respectively. The serum dilution is selected at an OD value of 1, and incubation is carried out at 37°C for 1 hour." The remaining steps are the same. Among them, the KLH-H-5F-1 immune group is competed with 5F-MDMB-PICA, the KLH-H-5F-2 immune group is competed with 5F-EMB-PICA, and the KLH-H-4F immune group is competed with 4F-MDMB-BUTICA. The chemical formulas of 4F-MDMB-BUTICA, 5F-MDMB-PICA, and 5F-EMB-PICA are as follows:

[0097] The results are shown in Figure 11. The IC values ​​of the antibodies produced after immunization with the three groups of antigens for their respective analytes were compared. 50 It was found that the antibodies produced by the hapten H-4F had an IC of 4F-MDMB-BUTICA. 50 The value is the lowest, while the hapten H-5F-1 and H-5F-2 produce antibody IC 50 The values ​​were all above 100 μM, indicating that the antibodies produced by H-4F were more specific. Therefore, mice immunized with H-4F were subsequently selected for hybridoma fusion to prepare monoclonal antibodies.

[0098] Example 7 Preparation of Synthetic Cannabinoid Monoclonal Antibodies (Originated by Fusion of Spleen Cells from Mice Immunized with KLH-H-4F)

[0099] Preparation of immune spleen cells: The antibody titers and specificities of the serum of mice immunized with three groups of immune antigens (KLH-H-5F-1, KLH-H-5F-2, and KLH-H-4F) were evaluated by ELISA. 50 The value was the smallest (the minimum IC value of 4F-MDMB-BUTICA in mice immunized with KLH-H-4F was determined by competitive ELISA). 50 The mice in the KLH-H-4F immunization group (with a KLH-H-4F value of 26.05 μM) were given a final boost immunization three days before fusion, i.e., 150 μg of the immune antigen, i.e., KLH-H-4F, 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.

[0100] 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.

[0101] 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.

[0102] Hybridoma Screening and Monoclonalization: On the second day after cell fusion, the cell fusion assay was checked for contamination by microscopically observing the bottom of the cell plate for contaminants. On the third day after fusion, the cells were half-changed with HAT complete medium. On days 5-7 after fusion, depending on cell growth status, the cells were half-changed with HT complete medium. The change procedure was the same as on the third day, i.e., half of the original medium was aspirated and an equal amount of fresh medium was added. After 7 days, the cells were fully changed to prevent the impact of specific antibodies secreted by unfused splenocytes on the test results. On the second day after the full change, a small amount of cell culture supernatant was diluted and screened for positive wells using indirect ELISA. Positive hybridoma cells were screened by competitive ELISA with 400 nM of the target molecule synthetic cannabinoid. Wells with an inhibition rate of 90% at 400 nM synthetic cannabinoid were selected and monocloned by limiting dilution until a monoclonal cell line stably expressing synthetic cannabinoid-specific antibodies was obtained. This cell line was expanded and cryopreserved for use in subsequent experiments.

[0103] Example 8: Large-Scale Preparation (In Vivo Induction Method) and Purification of Monoclonal Antibodies (mAb-4F)

[0104] Ten six-week-old female Balb / c mice were intraperitoneally injected with 500 μL of liquid paraffin per mouse. One week later, one million hybridoma cells were injected into each mouse to induce peritoneal tumors and ascites. One week later, the abdomen of the mice was significantly enlarged. The mice were sacrificed by cervical dislocation, and the abdominal fluid was removed by incision. After cryocentrifugation, the supernatant and lower sediment were discarded, and the cells were aliquoted and frozen at -80°C until use. The collected ascites solution was diluted 3 times with equilibrated wash buffer and then loaded onto a Protein A affinity column (Changzhou Tiandi Renhe Biotechnology Co., Ltd.). This was repeated 3 times. The impurities were then eluted from the affinity column with 5 column volumes of equilibrated wash buffer (0.15M NaCl, 20mM Na2HPO4, pH 7.0). The antibody was then eluted from the affinity column by adding 2 mL of elution buffer (0.1M glycine, pH 2.7) each time. 200 μL of neutralizing buffer (1M Tris-HCl, pH 8.5) was added to each collection tube in advance to neutralize the antibody solution. The eluted solution was concentrated using a 50kD ultrafiltration tube (Millopore) and stored in PBS. After concentration was determined, 10 mg / mL aliquots were frozen at -80°C until use.

[0105] Example 9 Isothermal Titration Calorimetry (ITC) Determination of the Affinity of Monoclonal Antibody mAb-4F to Synthetic Cannabinoids

[0106] Isothermal titration calorimetry (ITC) experiments were performed using a MICROCAL PEAQ-ITC (Malvern, UK). In all experiments, 4F-MDMB-BUTICA (150 μM) was dissolved in PBS + 1% DMSO. At 25°C, 80 μL of the synthetic cannabinoid solution (150 μM 4F-MDMB-BUTICA) was titrated into 10 μM mAb-4F at 120 s intervals. The binding data were processed using ITC-Analysis software, corrected for heat of dilution, fitted with a single-site binding model, and calculated Kd, binding parameter N, and ΔH. As shown in Figure 13, mAb-4F had significant binding affinity for 4F-MDMB-BUTICA, with a Kd value of 4.56 nM, a binding parameter N of 1.47, and a ΔH of -9.07 ± 0.118 kcal / mol.

[0107] Example 10 Antibody Amino Acid Sequence Determination

[0108] The antibody amino acid sequence was determined by hybridoma cell gene sequencing. The monoclonal cell line with high affinity to 4F-MDMB-B UTICA was screened and 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 to rapidly produce antibodies in large quantities in a eukaryotic expression system, comprising a mAb-4F light chain and a mAb-4F heavy chain. The amino acid sequence of the mAb-4F light chain variable region is shown in SEQ ID NO.1: DVVMTQTPLSLPVSLGDQASISCRSSQSLVLSNGNTFLHWYLQKSGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKITRVEAEDLGVYFCCQSTHVPWTFGGGTKLEIK; the amino acid sequence of the mAb-4F heavy chain variable region is shown in SEQ ID NO.2: DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYITYDGTNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCARVGTNWDDWGQGTTLTVSS.

[0109] The nucleotide sequence encoding the amino acid sequence of the mAb-4F light chain variable region as shown in SEQ ID NO.1 is shown in SEQ ID NO.3: GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACTCAGTAATGGAAACACCTTTTTACATTGGTACCTGCAGAAGTCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCCGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCACTAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTGTCAAAGTACACATGTCCCGTGGACGTTCGGTGGAGGTACCAAACTGGAAATCAAA; the nucleotide sequence encoding the amino acid sequence of the mAb-4F heavy chain variable region as shown in SEQ ID NO.2 is shown in SEQ ID Shown in NO.4: GATGTACAGCTTCAGGAGTCAGGACCTGGCCTCGTGAAAACCTTCTCAGTCTCTGTCTCTCACCTGCCTTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATAACCTACGACGGTACC AATAACTACAACCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGACTACTGAGGACACAGCTACATATTACTGTGCAAGGGTAGGAACTAACTGGGACGACTGGGGCCAGGGCACCACTCTCACAGTCTCCTCA.

[0110] Example 11 Establishment of a competitive ELISA method based on mAb-4F monoclonal antibody

[0111] The affinity of the ELISA method was improved by coating with heterologous proteins. After screening the coating antigens of various structural analogs, including BSA-H-4F, BSA-H-5F-1, BSA-H-5F-2, and BSA-H08, the use of BSA-H08 significantly improved the detection ability of mAb-4F. Therefore, BSA-H08 was used as the coating antigen for mAb-4F detection in subsequent experiments.

[0112] The specific steps of the competitive ELISA method are as follows:

[0113] Coating: Dilute the coating antigens BSA-H-4F, BSA-H-5F-2, and BSA-H08 to 1 μg / ml in coating solution, add 100 μl / well to a 96-well ELISA plate, seal with film, and incubate overnight at 4°C. The coating solution formula is as follows: KCl 0.2g, KH2PO4 0.24g, Na2HPO4.12H2O 3.63g, NaCl 8g, and dilute to 1000mL (pH 7.4) with ddH2O.

[0114] Blocking: Wash the plate three times with washing buffer, add 100 μl / well of blocking buffer, and incubate at 37°C for 1.5 h. The blocking buffer formula is as follows: KCl 0.2 g, KH2PO4 0.24 g, Na2HPO4.12H2O 3.63 g, NaCl 8 g, 50 g skim milk powder, and dilute to 1000 mL (pH 7.4) with ddH2O.

[0115] Primary antibody incubation: After blocking, wash the plate five times with washing buffer, add 50 μL of 4F-mAb monoclonal antibody (OD value is 1 at this dilution) and 50 μL of different concentrations of 4F-MDMB-BUTICA solution (250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, 0 nM) to the wells, and incubate at 37°C for 1 h;

[0116] Secondary antibody incubation: add 100 μL / well of goat anti-mouse HRP enzyme-labeled secondary antibody diluted 1:5000 and incubate at 37°C for 30 min;

[0117] Color development: 50 μL / well of TMB color development solution (Biyuntian, P0209 product) was added and incubated in the dark for 10 min;

[0118] 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.

[0119] The preparation method of BSA-H08 is as follows:

[0120] 1. Preparation of hapten compound H08:

[0121] Chemical structure of H08:

[0122] Synthesis route:

[0123] 1H-indazole-3-carboxylic acid (500 mg, 1 eq) and L-tert-leucine methyl ester hydrochloride (673 mg, 1.2 eq) were subjected to a condensation reaction in the presence of carbodiimide hydrochloride EDCI (887 mg, 1.5 eq), 1-hydroxybenzotriazole HOBt (625 mg, 1.5 eq) and N,N-diisopropylethylamine DIPEA (1196 mg, 3 eq) to produce intermediate 3; intermediate 3 (500 mg, 1 eq) was subjected to a substitution reaction with 1-bromobutane (285 mg, 1.2 eq) in the presence of NaH (83 mg, 2 eq) to produce intermediate 4; intermediate 4 was hydrolyzed and demethylated under alkaline conditions to produce intermediate 5 with a carboxyl group. 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 obtain a hapten precursor, which was then hydrolyzed under alkaline conditions to obtain the synthetic cannabinoid hapten compound H08. 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). 13C 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.

[0124] 2. Preparation of coating antigen BSA-H08:

[0125] Chemical structure:

[0126] 5 mg of hapten compound H08 was weighed separately, EDCI (6 eq), NHS (6 eq), Et3N (6 eq) and hapten compound H08 were dissolved in 500 μL DMF, and stirred at room temperature for 6 h until the hapten reaction was complete. BSA PBS solution (10 mg BSA protein was weighed and dissolved in PBS solution to 5 mg / mL) was added and stirred at 4°C overnight. The next day, the mixture was taken out and placed in a MW 3000 dialysis bag, and dialyzed against 500 mL PBS (pH 7.4) each time for a total of 4 times to obtain the coated antigen BSA-H08.

[0127] IC values ​​of mAb-4F and 4F-MDMB-BUTICA under different coating protein conditions 50 The assay was performed by adding 50 μL mAb-4F (OD value is 1 at this dilution) and 50 μL 4F-MDMB-BUTICA solution (250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, 0 nM) during the primary antibody incubation of the above ELISA method.

[0128] The results are shown in Table 1:

[0129] Table 1

[0130] It can be seen that the use of BSA-H08 can improve the detection effect several times. Therefore, when mAb-4F was used in 4F-MDMB-BUTICA detection in the later stage, BSA-H08 was used as the coating antigen.

[0131] The specific steps of the competitive ELISA method are as follows:

[0132] Coating: Dilute the coating antigen BSA-H08 to 1 μg / ml in 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.

[0133] Blocking: Wash the plate three times with washing buffer, add 100 μl / well of blocking buffer, and incubate at 37°C for 1.5 h. The blocking buffer formula is as follows: KCl 0.2 g, KH2PO4 0.24 g, Na2HPO4.12H2O 3.63 g, NaCl 8 g, 50 g skim milk powder, and dilute to 1000 mL (pH 7.4) with ddH2O.

[0134] Incubation with primary antibody: Wash the plate 5 times with washing buffer, add 50 μl of 167 ng / mL synthetic cannabinoid monoclonal antibody mAb-4F and 50 μl of PBS solution containing different concentrations of 4F-MDMB-BUTICA (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 4F-MDMB-BUTI CA test sample solutions (urine and hair samples), where the concentrations of 4F-MDMB-BUTICA in urine samples were 0, 0.14, 0.41, 1.23, 3.70, 11.11, 33.33, and 100 ng / mL; and the concentrations of 4F-MDMB-BUTICA 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;

[0135] 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;

[0136] Color development: 50 μL / well of TMB color development solution (Biyuntian, P0209 product) was added and incubated in the dark for 10 min;

[0137] 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.

[0138] Among them, urine pretreatment steps: artificial urine (Shanghai Yuanye Biotechnology Co., Ltd.) was adjusted to pH 7.4 using 1M NaOH solution, and added to a 96-well ELISA plate containing mAb-4F with or without 4F-MDMB-BUTICA, and incubated for detection.

[0139] Hair pretreatment steps: 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 (Zhejiang Noga Biotechnology Co., Ltd.), with or without the addition of 4F-MDMB-BUTICA. After grinding in a hair grinder for 10 minutes, centrifuge the supernatant, add it to a 96-well ELISA plate containing mAb-4F, and incubate for detection.

[0140] When the synthetic cannabinoid monoclonal antibody mAb-4F and PBS solutions containing different concentrations of 4F-MDMB-BUTICA were added to the wells, the 4F-MDMB-BUTICA calibration curve was established as shown in Figure 12. The IC values ​​of the synthetic cannabinoid antibody mAb-4F and 4F-MDMB-BUTICA were 50 The value is 2.108ng / mL.

[0141] The competitive ELISA method for determining 4F-MDMB-BUTICA in urine and hair samples (the wells were added with the synthetic cannabinoid monoclonal antibody mAb-4F and the test sample solutions (urine and hair samples) containing different concentrations of 4F-MDMB-BUTICA, respectively) was established. The standard curve is shown in Figure 14. In the competitive ELISA, the detection limits for urine were 0.137 ng / mL and the detection limits for hair were 0.012 ng / mg, respectively.

[0142] Example 12 Establishment of colloidal gold immunochromatography method based on mAb-4F monoclonal antibody

[0143] Step 1: H08-BSA antigen (0.8 mg / mL) and goat anti-mouse secondary antibody (Jackson ImmunoResearch, 205-035-108) (0.4 mg / mL) were streaked onto NC membrane using a gold streaker at a rate of 1 μL / cm and dried at 37°C. These served as the test line (T) and quality control line (C), respectively.

[0144] Step 2: Assemble the sample pad, NC membrane, and absorbent pad on the PVC base plate in order, and use a chopper to cut into 3mm wide test strips;

[0145] Step 3: 50 μL of PBS solution containing different concentrations of 4F-MDMB-BUTICA, urine and hair sample processed solutions, and 50 μL of colloidal gold solution modified with antibody mAb-4F (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. Under the action of capillary siphon, the mixed solution moved along the chromatography direction. When it reached the test line T and the quality control line C, the ratio of the test line to the quality control line was read by the colloidal gold reader TSR-100 (Hangzhou Aosheng Instrument Co., Ltd.). According to the different 4F-MDMB-BUTICA concentrations, the different values ​​of the test line T and the quality control line C were obtained. The ratio T / C of the test line and the quality control line was obtained. A standard curve was made by using this ratio and the corresponding 4F-MDMB-BUTICA concentration to detect this synthetic cannabinoid. Among them, the concentrations of 4F-MDMB-BUTICA in urine samples were 0, 0.5, 1, 2, 5, 10, 20, and 50 ng / mL; the concentrations of 4F-MDMB-BUTICA in hair samples were 0, 0.05, 0.1, 0.2, 0.5, 1, 2, and 5 ng / mg.

[0146] Preparation of colloidal gold solution modified with mAb-4F: 2 mL of 1% trisodium citrate solution was quickly added to 100 mL of boiling 0.01% HAuCl₄ solution. The solution was stirred and boiled for 5 minutes. The colloidal gold solution was then cooled to room temperature and stored at 4°C. mAb-4F was attached to the colloidal gold using electrostatic adsorption. First, 1 mL of colloidal gold solution was pipetted into a 1.5 mL EP tube. The pH of the colloidal gold solution was adjusted to 8.2 with 0.1 mol / L K₂CO₃. Then, 8 μg of the synthetic cannabinoid antibody mAb-4F was added and incubated at room temperature for 1 hour under rotating rotation. Bovine serum albumin (BSA) dissolved in 0.01 M boric acid buffer (BB, pH 8.2) to a final concentration of 10 mg / mL was added to the mixture and incubated for an additional hour. After centrifugation at 10,000 rpm / min for 15 minutes, the precipitate was resuspended in 100 μL of BB solution containing 2% bovine serum albumin and 3% sucrose to obtain the gold-labeled antibody stock solution.

[0147] The pretreatment steps for the colloidal gold immunochromatographic assay for urine and hair samples were the same as those in Example 11. The results are shown in Figure 15 , where A, C, and E are colloidal gold test strip images for PBS, urine, and hair samples, respectively, and B, D, and F are numerical graphs of the colloidal gold test strip images for PBS, urine, and hair samples, respectively. The abscissa represents concentration, and the ordinate represents T / C. As the concentration of 4F-MDMB-BUTICA increases, the T / C value gradually decreases, indicating that the established colloidal gold test strip immunochromatographic assay is capable of quantifying 4F-MDMB-BUTICA in urine and hair.

[0148] Example 13 HEK 293F cells expressing 4F-MDMB-BUTICA monoclonal antibody

[0149] 1. Plasmid construction

[0150] The full-length expression plasmids of the light and heavy chains of the 4F-MDMB-BUTICA specific monoclonal antibody mAb-4F were constructed respectively: 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 signal peptide + light chain variable region or heavy chain variable region + mouse constant region (IgG1 constant region and kappa constant region) were connected to the pcDNA3.4 vector, and the connection method was shown in Figure 16. The gene synthesis was completed by Detai Biotechnology (Nanjing) Co., Ltd., and the obtained light and heavy chain plasmids are shown in Figures 17 and 18. 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 murine kappa sequence is shown below: SEQID NO.8: AGGGCCGACGCAGCTCCTACCGTGTCTATCTTCCCCCCTAGCAGCGAGCAGCTGACATCAGGAGGAGCTAGCGTGGTCTGCTTCCTGAACAACTTCTACCCCAAGGACATCAACGTCAAGTGGAAGATCGACGGCAGCGAGAGGCAGAACGGCGTGCT GAACTCTTGGACCGACCAGGATAGCAAGGACAGCACCTACAGCATGAGCAGCACCCTGACCCTGACCAAGGACGAGTACGAGCGGCACAACAGCTACACCTGCGAGGCTACACACAAGACCAGCACCAGCCCCATCGTGAAGAGCTTCAACCGGAACGAGTGC.

[0151] 2. Plasmid transformation

[0152] 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.

[0153] The full-length expression plasmids of mAb-4F light and heavy chains 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.

[0154] 3. Plasmid Extraction

[0155] Sequence-verified mAb-4F full-length expression plasmids for the light and heavy chains were removed from a −80°C freezer and placed in LB medium containing 1% ampicillin (Shanghai Beyotime Biotechnology Co., Ltd.). The cells were shaken at 180 rpm at 37°C for 14 h. The cells were removed the next day and operated using a plasmid extraction kit (Kangwei Century Biotechnology Co., Ltd.). The plasmid concentrations were measured using a NanoDrop assay; the extracted light chain plasmid concentrations were 573.431 ng / μL, and the heavy chain plasmid concentrations were 584.859 ng / μL. The cells were then frozen in a −20°C freezer until use.

[0156] 4. Cell culture

[0157] 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.

[0158] 5. Cell transfection

[0159] When the density of HEK 293F cells in 300 mL reaches 3×10 6 When the cell culture medium reached 400 cells / mL, transfection was initiated. 150 μg each of the full-length light and heavy chain expression plasmids for mAb-4F were added to 10 mL of blank culture medium. 1.2 mg of 1 mg / mL PEI transfection reagent (PolySciences) was slowly added dropwise. After vortexing and allowing to stand for 15 minutes, the mixture was slowly added to the cell culture medium. Five days after transfection, the cell supernatant was collected for purification.

[0160] 6. Antibody Purification

[0161] 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 8.

[0162] 7. Validation of eukaryotic expression antibodies

[0163] The operation steps are the same as those in Example 11. The specific competitive concentrations of 4F-MDMB-BUTICA are 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, and 0 ng / mL. The results are shown in FIG17 . The IC values ​​of mAb-4F expressed and purified by the eukaryotic system and 4F-MDMB-BUTICA are 50The value was 1.502 ng / mL. The results showed that the affinity of the antibody prepared by hybridoma cell ascites was similar to that of 4F-MDMB-BUTICA, indicating that the plasmid can correctly express the specific antibody in the eukaryotic system.

[0164] Example 14 mAb-4F Specificity

[0165] Specificity analysis was performed using a competitive ELISA method with the same operating steps as in Example 11. The competitive concentrations of synthetic cannabinoids were 200, 66.67, 22.22, 7.41, 2.47, 0.82, 0.27, and 0 ng / mL, and the IC 50 IC values ​​of monoclonal antibodies and various synthetic cannabinoids 50 The values ​​are shown in Table 2. 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.

[0166] Table 2 mAb-4F and IC of synthetic cannabinoids 50 value

[0167] The results indicate that the monoclonal antibody mAb-4F 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 The general structural formula of the compound 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 The hydrocarbon group, p-phenyl, m-phenyl, o-phenyl; R1 is substituted or unsubstituted C6-C 10 Aryl, substituted or unsubstituted C3-C 10 of hydrocarbon groups; R2 is a single bond, a double bond, a triple bond, a heteroatom / halogen substituted or unsubstituted alkyl group, a substituted or unsubstituted heterocyclic group containing 1 to 3 heteroatoms / halogens, a methyl group or an ethyl group.

2. The synthetic cannabinoid hapten compound according to claim 1, characterized in that The structural formula of the compound 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 obtained 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 any one of 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 any one of claims 6 to 7.

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 4F-MDMB-BUTICA, ADB-BUTINACA, ADB-CHMICA, AB-4en-PINACA, ADB-4en-PINACA, 5F-AMB-PICA, 5F-EMB-PICA, 5F-MDMB-PICA, 5F-MPP-PICA, 5F-APP-PICA, 5F-EDMB-PICA, 5Br-AMB-PICA, 4Cl-MDMB-PICA, AMB-4en-PICA, AMB-CHMICA, MDMB-4en-PICA, MPP-PICA, MDMB-4en-PINACA butanoic acid metabolite, MDMB-BUTINACA butanoic acid metabolite, 5-fluoro MDMB-PICA metabolite 7, 4F-MDMB-BUTICA butanoic acid metabolite or 4-fluoro MDMB-BINACA 3,3-dimethylbutanoic acid.

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 described in claim 12.

Images