A detection method and detection kit for synthetic cannabinoids

Abstract

The present invention relates to the field of biotechnology, in particular to the field of IPC G01N33, and more specifically to a method for detecting synthetic cannabinoids and a detection kit. The present application independently developed the hapten, complete antigen and antibody of the synthetic cannabinoid Natasha, and used colloidal gold to prepare a synthetic cannabinoid detection kit. The prepared kit is convenient for detection, fast in detection speed, high in sensitivity and specificity, and has a good effect on the general screening of Natasha-type drugs, making up for the gap in the market for the lack of relevant detection kits for drugs such as Natasha. The cannabinoid detection kit prepared by the present application can be used for the general screening of drugs such as Natasha, providing effective technical means for front-line case handlers and having a certain application market.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to the field of IPC G01N33, and more specifically to a detection method and a detection kit for synthetic cannabinoids. Background Art

[0002] According to the World Drug Report 2019, synthetic cannabinoids are the fastest growing new psychoactive substances, and their trafficking and abuse have become a global problem.

[0003] Synthetic cannabinoids (SCs) are a class of cannabinoids that have similar pharmacological and physiological effects to delta-9-Tetrahydrocannabinol (TETRAHYD 9 -THC)-like compounds. Since the emergence of the first generation of illegal JWH SCs products in 2006, SCs have gradually become one of the most widely abused new psychoactive substances in the world. This type of substance mainly binds to the cannabis receptors CB1 or CB2, thereby producing physiological and pharmacological effects similar to or even stronger than tetrahydrocannabinol in the body. It was originally synthesized by scientists for the purpose of treating pain, and then developed into a third-generation drug, i.e. a new psychoactive substance, by an underground drug factory. As a substitute for natural cannabis, this type of substance has many characteristics such as long duration of action, ease of use, and easy availability. The main users are young people aged 12 to 20.

[0004] Due to the illegal abuse of SCs in recent years, the number of emergency cases caused by this type of drug has continued to increase, and it is related to many severe poisoning and death cases around the world. In view of this, countries around the world have taken measures. In March 2011, the US Drug Enforcement Administration listed JWH, 018, JWH, 073, JWH, 200, CP-47, 497 and CP-47, 497-C8 as Class I controlled drugs. In March 2013, another three SCs UR144, XLR-11 and AKB-48 were listed as Class I controlled drugs. In 2014, 81 new psychoactive substances were included in the early warning system detection by the European Union, of which 36% were SCs. In the "Catalogue of Psychotropic Drugs (2013 Edition)" implemented on January 1, 2014, my country listed 12 SCs such as JWH-018, JWH-250, and JWH-073 as Class I psychotropic drugs. If humans inhale SCs in large quantities, it will cause high blood pressure, anxiety, numbness, epilepsy, slurred speech and other poisoning symptoms, which seriously endanger human health. On March 15, 2021, the entire series of synthetic cannabinoids will be regulated.

[0005] There are more than 600 types of SCs reported so far, and there are many ways to classify them. The United Nations Office on Drugs and Crime (UNODC) mainly divides synthetic cannabinoids into classic cannabis, naphthoyl indoles, phenylacetyl indoles, benzoyl indoles, naphthylmethyl indoles, adamantane formyl indoles, naphthoyl pyrroles, cyclopropyl formyl indoles, indazole amides and other synthetic cannabinoids based on their chemical structure. Most synthetic cannabinoids are lipophilic and non-polar compounds, generally composed of 20 to 26 carbon atoms. The typical structure of synthetic cannabinoids mainly consists of four parts: a parent nucleus with substituents, a linking part, a linking group and a terminal substituent. The linking group refers to the chemical bond between the pentyl indole ring or the indazole ring and the naphthalene ring, mainly a carbonyl group, an ester bond, and an amide bond.

[0006] Natasha is a type of synthetic cannabinoid. This new type of drug is made of various petals or roots, stems and leaves of plants into tobacco, which contains this type of synthetic cannabinoid, which is extremely harmful. Its main ingredients are ADB-FUBINACA, AMB-FUBINACA and 5F-ADB. The Ministry of Public Security, the National Health Commission and the State Food and Drug Administration listed them in the supplementary catalog of non-medical narcotic drugs and psychotropic drugs on September 1, 2018.

[0007]

[0008] In the prior art, a patent document with application publication number CN 101580544A discloses a colloidal gold labeled cannabis and tetrahydrocannabinol monoclonal antibody immunoassay plate. The immunoassay plate prepared by the colloidal gold method can detect tetrahydrocannabinol in human specimens such as drugs or urine samples. The detection speed is fast, the sensitivity is high, and the specificity is strong. However, the detection effect on Natasha-type synthetic cannabinoids is limited.

[0009] The patent document with application publication number CN110161141A discloses a method for detecting indazole derivatives. Through gas chromatography-mass spectrometry (GC-MS) technology, indazole amide synthetic cannabinoids can be qualitatively and quantitatively analyzed. However, the method is relatively complicated, and the detection time is long and the detection cost is high.

[0010] At present, the main method for detecting synthetic cannabinoids is GC-MS. Although it has advantages in accuracy and sensitivity, the detection cost is high and the time is long. There is no detection kit for Natasha-type synthetic cannabinoids on the market. Summary of the invention

[0011] In order to solve the above problems, the first aspect of the present invention provides a detection kit for synthetic cannabinoids, which includes a reagent strip and a plastic part.

[0012] Preferably, the reagent strip comprises a backing plate, a sample pad, a colloidal gold binding pad, a coating membrane and a water absorbent pad.

[0013] Preferably, the preparation method of the detection kit is: sticking the sample pad, colloidal gold binding pad, coating membrane and water absorbent pad on the backing plate in order; cutting the stuck plate into 3 mm wide strips, assembling the kit, and then putting it into an aluminum foil bag with a desiccant and sealing it for storage.

[0014] Preferably, the colloidal gold binding pad is prepared by adsorbing the synthetic cannabinoid colloidal gold labeled antibody onto glass fiber and drying the resulting mixture.

[0015] The drying temperature is 35-40°C; more preferably, 37°C.

[0016] Preferably, the raw materials for preparing the synthetic cannabinoid colloidal gold-labeled antibody include gold label diluent and synthetic cannabinoid antibody gold label solution.

[0017] Preferably, the weight ratio of the synthetic cannabinoid antibody gold-labeled solution to the gold-labeled diluent is 1: (20-200).

[0018] Preferably, the gold label diluent is an aqueous solution of one or more of Tris (tris(hydroxymethylaminomethane), BSA (bovine serum albumin), sucrose, trehalose, and TritonX-100 (polyethylene glycol octylphenyl ether); further preferably, the gold label diluent is an aqueous solution of Tris, BSA, sucrose, trehalose, and TritonX-100.

[0019] Preferably, the gold label diluent is 0.5-1.0wt% Tris + 0.8-1.5wt% BSA + 5-15wt% sucrose + 2-8wt% trehalose + 0.1-0.8vol% TritonX-100 + water as the remainder; further preferably, the gold label diluent is 0.848wt% Tris + 1.00wt% BSA + 10wt% sucrose + 5wt% trehalose + 0.4vol% TritonX-100 + water as the remainder.

[0020] In some preferred schemes, the gold label diluent is 0.5-1.0wt% Tris+0.8-1.5wt% BSA+5-15wt% sucrose+2-8wt% trehalose+0.1-0.8vol% TritonX-100+water surplus, which can improve the stability and specificity of the detection kit. This may be due to the synergistic effect of BSA and other raw materials in the raw materials, which can protect the labeled protein and prevent the protein from denaturing during the drying process, while also playing a certain blocking role on the non-specific reaction between proteins, and the addition of an appropriate amount of sucrose can also alleviate the adsorption between the colloidal gold label and the colloidal gold binding pad, thereby reducing the problem of difficulty in releasing the colloidal gold label after moisture absorption, thereby improving the stability of the kit, but its improvement on stability is limited. The applicant unexpectedly found that adding a certain amount of trehalose can further improve the problem of stability of the colloidal gold label, but its cost is relatively high.

[0021] Preferably, the preparation method of the synthetic cannabinoid antibody gold-labeled solution is: mixing the colloidal gold solution with the synthetic cannabinoid antibody and centrifuging to obtain the solution.

[0022] Preferably, the preparation method of the synthetic cannabinoid antibody gold label solution is: adjust the pH of the colloidal gold solution with 0.1M potassium carbonate to a slightly alkaline isoelectric point of the antibody to be labeled, add the synthetic cannabinoid antibody and 10% BSA, and stir the reaction for 30-60 minutes. Collect the precipitate after centrifugation. The isoelectric points of common proteins to be labeled are shown in the following table:

[0023] Protein to be labeled Isoelectric point Protein to be labeled Isoelectric point IgG by affinity chromatography 7.6-8.0 Avidin 10.0 Monoclonal antibodies 8.2-8.5 Streptavidin 6.6 IgG 9.0 ConA 8.0

[0024] In some preferred embodiments, the protein to be labeled for the synthetic cannabinoid antibody in the present application is IgG or monoclonal antibody for affinity chromatography.

[0025] Preferably, the preparation method of the colloidal gold solution is trisodium citrate reduction method. This method is a classic method for preparing colloidal gold. First, a certain amount of 0.01% HAuCl4 solution is heated to boiling, and an appropriate amount of 1% trisodium citrate aqueous solution is quickly added. The color first turns blue and then red. Continue to boil until a transparent orange-red color appears. Turn off the heater, cool naturally to room temperature, and then add purified water to add the colloidal gold solution to 100 ml to prepare colloidal gold solutions of different diameters.

[0026] The colloidal gold preparation of various particles is detailed in the table below.

[0027]

[0028] Preferably, the synthetic cannabinoid antibody is a polyclonal antibody or a monoclonal antibody.

[0029] The polyclonal antibodies are AMB-FUBINACA rabbit anti-polyclonal antibody and 5F-ADB rabbit anti-polyclonal antibody.

[0030] The monoclonal antibodies are AMB-FUBINACA mouse anti-monoclonal antibody and 5F-ADB mouse anti-monoclonal antibody.

[0031] Preferably, the method for preparing the polyclonal antibody is: immunizing New Zealand white rabbits with synthetic cannabinoid complete antigen 2#, with an immunization dose of 0.8-1.2 mg / time, and multiple subcutaneous injections on the back; further preferably, the method for preparing the polyclonal antibody is: mixing synthetic cannabinoid complete antigen 2# with Freund's complete adjuvant (FCA) and fully emulsifying to obtain reagent 1, injecting reagent 1 into New Zealand white rabbits to complete the first immunization; mixing synthetic cannabinoid complete antigen 2# with Freund's incomplete adjuvant (FIA) and fully emulsifying to obtain reagent 2, and three weeks after the first immunization, injecting reagent 2 into New Zealand white rabbits for booster immunization, continuously boosting immunization 4-5 times, with an interval of 2-3 weeks between each booster immunization, and measuring its titer by ELISA method 10-15 days after the last booster immunization, and the titer reaches 10 5 At the above time, blood was collected and serum was separated and collected, and the obtained synthetic cannabinoid polyclonal antibodies were purified by affinity chromatography and frozen at -20°C for future use.

[0032] Preferably, the volume ratio of the synthetic cannabinoid complete antigen 2# to Freund's complete adjuvant or Freund's incomplete adjuvant is (1-2):(1-2); further preferably, the volume ratio is 1:1.

[0033] Preferably, the synthetic cannabinoid complete antigen 2# is AMB-FUBINACA complete antigen 2# or 5F-ADB complete antigen 2#.

[0034] Preferably, the coating membrane is prepared by spraying the test line (T line) reagent and the control line (C line) reagent on the nitrocellulose membrane respectively, and drying in an oven at 37° C. for 10 hours.

[0035] Preferably, the T line (detection line) is close to the sample pad, and the C line (control line) is close to the water absorbent pad.

[0036] Preferably, the spraying amount of the detection line reagent and the control line reagent is 0.5-2.0 μl / cm, respectively; more preferably, it is 1.0 μl / cm.

[0037] Preferably, the T-line reagent is a solution obtained by diluting synthetic cannabinoid complete antigen 1# with synthetic cannabinoid complete antigen 1# dilution solution.

[0038] Preferably, the concentration of the synthetic cannabinoid complete antigen 1# in the T-line reagent is 0.5-2.0 mg / mL.

[0039] Preferably, the synthetic cannabinoid complete antigen 1# dilution is one or more of 0.01M PBS buffer at pH 7.2-7.6, 1% BSA, 0.5% CaseinNa, and 1% Tritonx-100; further preferably, it is 0.01M PBS buffer at pH 7.2-7.6; further preferably, it is 0.01M PBS buffer at pH 7.4.

[0040] Preferably, the synthetic cannabinoid complete antigen 1# is AMB-FUBINACA complete antigen 1# or 5F-ADB complete antigen 1#.

[0041] Preferably, the C-line reagent is a solution obtained by diluting goat anti-rabbit antibody with goat anti-rabbit antibody diluent.

[0042] Preferably, the concentration of goat anti-rabbit antibody in the C-line reagent is 0.5-2.0 mg / mL.

[0043] Preferably, the goat anti-rabbit antibody diluent is one or more of 0.01M PBS buffer at pH 7.2-7.6, 1% BSA, 0.5% CaseinNa, 1% Tritonx-100; further preferably, it is 0.01M PBS buffer at pH 7.2-7.6; further preferably, it is 0.01M PBS buffer at pH 7.4.

[0044] In some preferred embodiments, the goat anti-rabbit antibody is purchased from Goat anti-Rabbit IgG sold by Hangzhou Longji Biotechnology Co., Ltd. with the product number PS01001.

[0045] Preferably, the preparation method of the synthetic cannabinoid complete antigen 2# is as follows: dissolve the hapten in an organic solvent, add a coupling agent, and stir for 2-6 hours to obtain a solution A; dissolve the carrier protein 2# in a buffer solution, drip into the solution A while stirring, continue stirring for 10-14 hours after dripping, and finally dialyze in a dialysate at low temperature for three days to obtain the solution. The chemical reaction formula is as follows: Fig.11 shown.

[0046] Preferably, the carrier protein 2# is one or more of bovine serum albumin (BSA), hemocyanin (KLH), chicken ovalbumin (OVA), human serum albumin, and hemoglobin; more preferably, it is KLH.

[0047] In some preferred embodiments, when preparing synthetic cannabinoid antibodies, the titer should reach 10 5When the above conditions are met, the synthetic cannabinoid antibodies prepared will have a certain specificity. However, the molecular weight of OVA is relatively low, and its immunogenicity is poor, making it difficult to prepare synthetic cannabinoid antibodies with higher titers. The applicant unexpectedly discovered that by using KLH as the carrier protein 2# to prepare the synthetic cannabinoid complete antigen 2#, synthetic cannabinoid complete antigen 2# can prepare synthetic cannabinoid antibodies with higher titers. Perhaps because the molecular weight of KLH exceeds 5000KDa and it comes from animals that are very different from humans, the titer and specificity of the synthetic cannabinoid antibodies prepared using it as raw material have been greatly improved, which can avoid detecting morphine, △ 9 -tetrahydrocannabinolic acid and other drugs, while accurately detecting ADB FUBINACA, AMBFUBINACA and 5F-ADB described in the present application, but it has no significant improvement in sensitivity.

[0048] Preferably, the hapten is AMB-FUBINACA hapten or 5F-ADB hapten.

[0049] Preferably, the organic solvent is one or more of N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF); more preferably, it is DMF.

[0050] Preferably, the coupling agent is one or more of N-hydroxysuccinimide, carbodiimide, formaldehyde, glutaraldehyde and the like; more preferably, it is N-hydroxysuccinimide (NHS) and EDC.

[0051] Preferably, the weight ratio of N-hydroxysuccinimide to carbodiimide is 1 (1-1.5); more preferably, it is 4:5.

[0052] In some preferred embodiments, the coupling agent is NHS and EDC, which can form stable esters while promoting the coupling of hapten and carrier protein, thereby improving the stability of the coupling product. The applicant unexpectedly found that when the weight ratio of N-hydroxysuccinimide to carbodiimide is 1: (1-1.5), the highest coupling efficiency can be obtained.

[0053] Preferably, the buffer is one or more of PBS buffer at pH 7.2 to 7.6, 0.5% CaseinNa, and 1% Tritonx-100; more preferably, it is PBS buffer at pH 7.2 to 7.6; and further preferably, it is PBS buffer at pH 7.4.

[0054] Preferably, the preparation method of the pH 7.4 PBS buffer is as follows: take 8.0 g NaCl, 0.2 g KH2PO4, 2.9 g Na2HPO4.12H2O, and 0.2 g KCl, add water to make up to 1 L, adjust the pH value to 7.4, and store at 4° C. for later use.

[0055] Preferably, the dialysate is a PBS buffer solution with a pH of 7.4.

[0056] Preferably, the weight ratio of the hapten to the carrier protein is 1:(2-20); further preferably, it is 1:5.

[0057] The applicant unexpectedly discovered that when preparing the synthetic cannabinoid complete antigen 2#, when the weight ratio of the hapten to the carrier protein is 1: (2-20), the immunogenicity and reactogenic properties of the antigen remain in a better state. This may be because the carrier protein not only imparts immunogenicity to the hapten, but also affects the memory characteristics of the hapten immune response. The specificity of the hapten is recognized by B cells, and the specificity of the carrier protein is recognized by T cells. Only the synergistic action of TB cells can initiate an antibody response to the hapten. The applicant also unexpectedly discovered that when preparing the synthetic cannabinoid complete antigen 1#, when the weight ratio of the hapten to the carrier protein is 1: (2-20), the sensitivity of the synthetic cannabinoid antibody-antigen binding is better. If the weight ratio of the hapten to the carrier protein is too high or too low, it will affect the competition and binding of the antibody and antigen, thereby affecting the performance of the detection kit.

[0058] Preferably, the preparation method of the synthetic cannabinoid complete antigen 1# is as follows: dissolve the hapten in an organic solvent, add a coupling agent, and stir for 2-6 hours to obtain a solution A; dissolve the carrier protein 1# in a buffer solution, drip into the solution A while stirring, continue stirring for 10-14 hours after dripping, and finally dialyze in a dialysate at low temperature for three days to obtain the solution. The chemical reaction formula is as follows: Fig.10 shown.

[0059] Preferably, the carrier protein 1# is one or more of bovine serum albumin (BSA), hemocyanin (KLH), chicken ovalbumin (OVA), human serum albumin, and hemoglobin; more preferably, it is BSA.

[0060] Preferably, the synthesis method of the AMB-FUBINACA hapten is as follows: 0.5 g AMB-FUBINACA is dissolved in 20 mL methanol, 2 mL 1 mol / L sodium hydroxide solution is added dropwise, and the mixture is stirred at room temperature overnight. After the reaction is complete, the solvent is evaporated under reduced pressure, water is added, the pH is adjusted to 3-4 with HCl, and the mixture is extracted with ether for several times. The organic phases are combined, dried with anhydrous magnesium sulfate, and the solvent is evaporated to obtain a white solid, which is the hapten. The chemical reaction formula is as follows: Figure 1 shown.

[0061] Preferably, the synthesis method of AMB-FUBINACA is as follows: 0.54 g of indole-1-(4-fluorobenzyl)-3-carboxylic acid, 1.09 g of Py-Bop and 10 mL of DMSO are placed in a three-necked flask, 1.05 g of L-valine methyl ester is added dropwise while stirring, and stirred at room temperature overnight. After the reaction is complete, it is poured into a sodium bicarbonate solution, extracted with ether, and purified by column chromatography to obtain a crystalline solid AMB-FUBINACA. The chemical reaction formula is as follows: Figure 2 shown.

[0062] Preferably, the synthesis method of indole-1-(4-fluorobenzyl)-3-carboxylic acid is as follows: 3.9 g of indole-1-(4-fluorobenzyl)-3-carboxylic acid methyl ester is dissolved in 50 mL of methanol, 20.6 mL of 1 mol / L sodium hydroxide solution is added dropwise, and stirred at room temperature overnight. After the reaction is complete, the solvent is evaporated under reduced pressure, the white solid is dissolved in water, the pH is adjusted to 3 with HCl, and extracted with ethyl acetate for multiple times, the organic phases are combined, dried with anhydrous magnesium sulfate, and the solvent is evaporated to obtain a white solid indole-1-(4-fluorobenzyl)-3-carboxylic acid. The chemical reaction formula is as follows Figure 3 shown.

[0063] Preferably, the synthesis method of indole-1-(4-fluorobenzyl)-3-carboxylic acid methyl ester is as follows: in an ice-water bath, 2.5 g of indole-3-carboxylic acid methyl ester, 1.75 g of potassium tert-butoxide and 100 mL of tetrahydrofuran are placed in a three-necked flask, stirred for 1 hour, and then 2.82 g of p-fluorobenzyl bromide is added, and stirred at room temperature overnight. After the reaction is complete, pour into 100 mL of water, extract with ethyl acetate for several times, combine the organic phases, dry with anhydrous magnesium sulfate, and evaporate the solvent to obtain colorless liquid indole-1-(4-fluorobenzyl)-3-carboxylic acid methyl ester. The chemical reaction formula is as follows Figure 4 shown.

[0064] Preferably, the synthesis method of indole-3-carboxylic acid methyl ester is as follows: in an ice-water bath, 9.0 g of indole-3-carboxylic acid and 150 mL of methanol are placed in a three-necked flask, and 9 mL of concentrated sulfuric acid is added dropwise. After the addition is complete, heat to vortex and react for 24 hours. After the reaction is complete, evaporate the solvent under reduced pressure, add ethyl acetate and water, extract several times, dry the organic phase with anhydrous magnesium sulfate, evaporate the solvent, a white solid precipitates, filter, and obtain white solid indole-3-carboxylic acid methyl ester. The chemical reaction formula is as follows Figure 5 shown.

[0065] Preferably, the synthesis method of the 5F-ADB hapten is as follows: 0.55g 5F-ADB is dissolved in 20mL methanol, 2.2mL 1mol / L sodium hydroxide solution is added dropwise, and stirred at room temperature overnight. After the reaction is complete, the solvent is evaporated under reduced pressure, water is added, the pH is adjusted to 3-4 with HCl, and the mixture is extracted with ether for several times, the organic phases are combined, dried with anhydrous magnesium sulfate, and the solvent is evaporated to obtain a white solid. The chemical reaction formula is as follows: Figure 6 shown.

[0066] Preferably, the synthesis method of 5F-ADB is as follows: 0.5 g of indole-1-(5-fluoropentyl)-3-carboxylic acid, 1.09 g of Py-Bop and 10 mL of DMSO are placed in a three-necked flask, 1.1 g of L-tert-leucine methyl ester is added dropwise while stirring, and stirred at room temperature overnight. After the reaction is complete, it is poured into a sodium bicarbonate solution, extracted with ether, and purified by column chromatography to obtain a crystalline solid. The chemical reaction formula is as follows: Figure 7 shown.

[0067] Preferably, the synthesis method of indole-1-(5-fluoropentyl)-3-carboxylic acid is as follows: 1.4 g of indole-1-(5-fluoropentyl)-3-carboxylic acid methyl ester is dissolved in 30 mL of methanol, 8 mL of 1 mol / L sodium hydroxide solution is added dropwise, and stirred at room temperature overnight. After the reaction is complete, the solvent is evaporated under reduced pressure, the white solid is dissolved in water, the pH is adjusted to 3-4 with HCl, and extracted with ethyl acetate for multiple times, the organic phases are combined, dried with anhydrous magnesium sulfate, and the solvent is evaporated to obtain a white solid indole-1-(5-fluoropentyl)-3-carboxylic acid. The chemical reaction formula is as follows Figure 8 shown.

[0068] Preferably, the synthesis method of indole-1-(5-fluoropentyl)-3-carboxylic acid methyl ester is as follows: in an ice-water bath, 2.0 g of indole-3-carboxylic acid methyl ester, 1.4 g of potassium tert-butoxide and 80 mL of tetrahydrofuran are placed in a three-necked flask, stirred for 1 hour, and then 2.02 g of 1-bromo-5-fluoropentane is added, and stirred at room temperature overnight. After the reaction is complete, pour into 100 mL of water, extract with ethyl acetate for several times, combine the organic phases, dry with anhydrous magnesium sulfate, and evaporate the solvent to obtain colorless liquid indole-1-(5-fluoropentyl)-3-carboxylic acid methyl ester.

[0069] The second aspect of the present invention provides a method for detecting synthetic cannabinoids, wherein 3 to 4 drops of a sample to be tested are dripped onto the synthetic cannabinoid detection kit prepared in the present application, and the test result is observed and determined for about 5 minutes.

[0070] If only one red line, line C, appears on the coating membrane, it indicates that the test result is positive. If both red lines, line C and line T, appear on the coating membrane, it indicates that the test result is negative. If the red line C does not appear on the coating membrane, it indicates that the test strip has expired.

[0071] Beneficial effects:

[0072] 1. The stability and specificity of the detection kit can be improved by selecting a gold standard diluent of 0.5-1.0wt% Tris+0.8-1.5wt% BSA+5-15wt% sucrose+2-8wt% trehalose+0.1-0.8vol% TritonX-100+water as the remainder.

[0073] 2. By selecting KLH as the carrier protein 2# to prepare the complete synthetic cannabinoid antigen 2#, the prepared synthetic cannabinoid antibody has high titer and good specificity of the kit.

[0074] 3. By selecting N-hydroxysuccinimide and carbodiimide in a weight ratio of 1 (1-1.5), the stability of the conjugate can be effectively improved while increasing the coupling degree.

[0075] 4. When preparing synthetic cannabinoid complete antigen 2# and synthetic cannabinoid complete antigen 1#, when the weight ratio of the hapten to the carrier protein is 1:(2-20), the immune effect and the sensitivity of the detection kit can be greatly improved.

[0076] 5. The present applicant independently developed the hapten, complete antigen and antibody of the synthetic cannabinoid Natasha, and used colloidal gold to prepare a synthetic cannabinoid detection kit. The prepared kit is convenient for detection, fast in detection speed, high in sensitivity and specificity, and has a good effect on the general screening of Natasha-type drugs, filling the gap in the market for the lack of detection kits for drugs such as Natasha.

[0077] 6. The cannabinoid detection kit prepared in this application can be used for general screening of drugs such as Natasha, providing effective technical means for front-line investigators and having a certain application market. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 is the synthetic chemical reaction formula of AMB-FUBINACA hapten;

[0079] Figure 2 is the synthetic chemical reaction formula of AMB-FUBINACA;

[0080] Figure 3 is the synthetic chemical reaction formula of indole-1-(4-fluorobenzyl)-3-carboxylic acid;

[0081] Figure 4 is the synthetic chemical reaction formula of indole-1-(4-fluorobenzyl)-3-carboxylic acid methyl ester;

[0082] Figure 5 is the synthetic chemical reaction formula of indole-3-carboxylic acid methyl ester;

[0083] Figure 6 is the synthetic chemical reaction formula of 5F-ADB hapten;

[0084] Figure 7 is the synthetic chemical reaction formula of 5F-ADB;

[0085] Figure 8 The synthetic chemical reaction formula of indole-1-(5-fluoropentyl)-3-carboxylic acid;

[0086] Fig. 9 is the synthetic chemical reaction formula of indole-1-(5-fluoropentyl)-3-carboxylic acid methyl ester;

[0087] Fig.10 The synthetic chemical reaction formula of the synthetic cannabinoid complete antigen 1# (hapten is AMB-FUBINACA hapten);

[0088] Fig.11 The synthetic chemical reaction formula of synthetic cannabinoid complete antigen 2# (hapten is AMB-FUBINACA hapten);

[0089] Fig.12 This is the NMR-H spectrum of the AMDFUBINACA hapten. DETAILED DESCRIPTION

[0090] Example

[0091] Example 1

[0092] Embodiment 1 provides a synthetic cannabinoid detection kit, which includes a reagent strip and a plastic part.

[0093] The reagent strip comprises a backing plate, a sample pad, a colloidal gold binding pad, a coating film and a water absorption pad.

[0094] The preparation method of the detection kit is as follows: a sample pad, a colloidal gold binding pad, a coating film and a water-absorbing pad are sequentially attached to a backing plate from bottom to top; the attached plate is cut into reagent strips with a width of not less than 3 mm, which are loaded into a plastic part, and then are loaded into an aluminum foil bag together with a desiccant and a small dropper for sealed storage.

[0095] The sample pad was purchased from Shanghai Hanyi Biotechnology Co., Ltd.

[0096] The backing plate is a PVC base plate.

[0097] The PVC base plate was purchased from Quzhou Maike New Materials Co., Ltd.

[0098] The absorbent pad was purchased from Shanghai Pharmaceuticals Group.

[0099] The preparation method of the colloidal gold binding pad is as follows: synthetic cannabinoid colloidal gold labeled antibody is adsorbed on glass fiber and dried at 37°C to obtain the colloidal gold binding pad.

[0100] The glass fiber was purchased from Tongsheng (Fuzhou) Paper Co., Ltd.

[0101] The raw materials for preparing the synthetic cannabinoid colloidal gold labeled antibody include gold labeling diluent and synthetic cannabinoid antibody gold labeling solution.

[0102] The weight ratio of the synthetic cannabinoid antibody gold-labeled solution to the gold-labeled diluent is 1:40.

[0103] The gold label diluent is 0.848 wt% Tris+1.00 wt% BSA+10 wt% sucrose+5 wt% trehalose+0.4 vol% TritonX-100+water as the remainder.

[0104] The preparation method of the synthetic cannabinoid antibody gold-labeled solution is as follows: adjust the pH of the colloidal gold solution to 8.0 with 0.1M potassium carbonate, add the synthetic cannabinoid antibody and 10% BSA, stir well to react for 30 minutes, and collect the precipitate after centrifugation.

[0105] The colloidal gold solution preparation method is trisodium citrate reduction method. This method is a classic method for preparing colloidal gold. First, 100 ml of 0.01% HAuCl4 solution is heated to boiling, and a certain amount of 1.5 ml of 1% trisodium citrate aqueous solution is quickly added. The color first turns blue and then red. Continue to boil until a transparent orange-red color appears. Turn off the heater, cool naturally to room temperature, and then add purified water to add 100 ml of colloidal gold solution to prepare a colloidal gold solution with a diameter of about 25 nm.

[0106] The synthetic cannabinoid antibody is a polyclonal antibody.

[0107] The polyclonal antibody is AMB-FUBINACA rabbit IgG antibody.

[0108] The preparation method of the polyclonal antibody is as follows: a synthetic cannabinoid complete antigen 2# is mixed with Freund's complete adjuvant (FCA) and fully emulsified to obtain reagent 1, and the reagent 1 is injected into New Zealand white rabbits to complete the first immunization; a synthetic cannabinoid complete antigen 2# is mixed with Freund's incomplete adjuvant (FIA) and fully emulsified to obtain reagent 2, and three weeks after the first immunization, the reagent 2 is injected into New Zealand white rabbits for booster immunization, and the booster immunization is continuously performed for 5 times, each booster immunization is separated by 3 weeks, and the titer is measured by ELISA method 15 days after the last booster immunization, and the titer reaches 10 5 When the time came, blood was collected and serum was separated and collected, which was purified by affinity chromatography to obtain synthetic cannabinoid polyclonal antibodies and stored at -20°C for future use.

[0109] The volume ratio of the synthetic cannabinoid complete antigen 2# to Freund's complete adjuvant or Freund's incomplete adjuvant is 1:1.

[0110] The synthetic cannabinoid complete antigen 2# is AMB-FUBINACA complete antigen 2#.

[0111] The coating membrane is prepared by spraying the test line (T line) reagent and the control line (C line) reagent on the nitrocellulose membrane respectively, and drying in an oven at 37° C. for 10 hours.

[0112] The nitrocellulose membrane was purchased from Sartorius.

[0113] The T line (test line) is close to the sample pad, and the C line (control line) is close to the absorbent pad.

[0114] The spraying amount of the test line reagent and the control line reagent is 1.0 μl / cm.

[0115] The T-line reagent is a solution obtained by diluting the synthetic cannabinoid complete antigen 1# with the synthetic cannabinoid complete antigen 1# diluent.

[0116] The concentration of synthetic cannabinoid complete antigen 1# in the T-line reagent is 1.0 mg / mL.

[0117] The synthetic cannabinoid complete antigen 1# dilution solution is a 0.01M PBS buffer solution at pH 7.4.

[0118] The synthetic cannabinoid complete antigen 1# is AMB-FUBINACA complete antigen 1#.

[0119] The C-line reagent is a solution obtained by diluting goat anti-rabbit antibody with goat anti-rabbit antibody diluent. The concentration of goat anti-rabbit antibody in the C-line reagent is 1.0 mg / mL.

[0120] The goat anti-rabbit antibody diluent is a 0.01M PBS buffer solution at pH 7.4.

[0121] The goat anti-rabbit antibody was purchased from Goat anti-Rabbit IgG sold by Hangzhou Longji Biotechnology Co., Ltd. with the catalog number PS01001.

[0122] The preparation method of the synthetic cannabinoid complete antigen 2# is as follows: dissolve the hapten in an organic solvent, add a coupling agent, and stir for 4 hours to obtain a solution A; dissolve the carrier protein 2# in a buffer solution, drip into the solution A while stirring, continue stirring for 12 hours after dripping, and finally dialyze in a dialysate at low temperature for three days to obtain the solution. The chemical reaction formula is as follows: Fig.11 shown.

[0123] The carrier protein 2# is KLH.

[0124] The hapten is AMB-FUBINACA hapten.

[0125] The organic solvent is DMF.

[0126] The coupling agents are N-hydroxysuccinimide (NHS) and electrolytic diimide (EDC).

[0127] The weight ratio of the N-hydroxysuccinimide to carbodiimide is 4:5.

[0128] The buffer solution is PBS buffer solution with a pH of 7.4.

[0129] The preparation method of the pH 7.4 PBS buffer is as follows: 8.0 g NaCl, 0.2 g KH2PO4, 2.9 g Na2HPO4.12H2O, and 0.2 g KCl are taken, water is added to make the volume to 1 L, the pH value is adjusted to 7.4, and stored at 4° C. for future use.

[0130] The dialysate is a PBS buffer solution with a pH of 7.4.

[0131] The weight ratio of the hapten to the carrier protein is 1:5.

[0132] The preparation method of the synthetic cannabinoid complete antigen 1# is as follows: dissolve the hapten in an organic solvent, add a coupling agent, and stir for 4 hours to obtain a solution A; dissolve the carrier protein 1# in a buffer solution, drip into the solution A while stirring, continue stirring for 12 hours after dripping, and finally dialyze in a dialysate at low temperature for three days to obtain the complete antigen. The chemical reaction formula is as follows: Fig.10 shown.

[0133] The carrier protein 1# is BSA.

[0134] The synthesis method of the AMB-FUBINACA hapten is as follows: 0.5 g AMB-FUBINACA is dissolved in 20 mL methanol, 2 mL 1 mol / L sodium hydroxide solution is added dropwise, and stirred at room temperature overnight. After the reaction is complete, the solvent is evaporated under reduced pressure, water is added, the pH is adjusted to 3-4 with HCl, and the mixture is extracted with ether for several times, the organic phases are combined, dried with anhydrous magnesium sulfate, and the solvent is evaporated to obtain a white solid. The chemical reaction formula is as follows: Figure 1 shown.

[0135] The synthesis method of AMB-FUBINACA is as follows: 0.54 g of indole-1-(4-fluorobenzyl)-3-carboxylic acid, 1.09 g of Py-Bop and 10 mL of DMSO are placed in a three-necked flask, 1.05 g of L-valine methyl ester is added dropwise while stirring, and stirred at room temperature overnight. After the reaction is complete, the mixture is poured into a sodium bicarbonate solution, extracted with ether, and purified by column chromatography to obtain crystalline solid AMB-FUBINACA. The chemical reaction formula is as follows: Figure 2 shown.

[0136] The synthesis method of indole-1-(4-fluorobenzyl)-3-carboxylic acid is as follows: 3.9 g of indole-1-(4-fluorobenzyl)-3-carboxylic acid methyl ester is dissolved in 50 mL of methanol, 20.6 mL of 1 mol / L sodium hydroxide solution is added dropwise, and the mixture is stirred at room temperature overnight. After the reaction is complete, the solvent is evaporated under reduced pressure, the white solid is dissolved in water, the pH is adjusted to 3 with HCl, and the mixture is extracted with ethyl acetate for multiple times. The organic phases are combined, dried with anhydrous magnesium sulfate, and the solvent is evaporated to obtain a white solid indole-1-(4-fluorobenzyl)-3-carboxylic acid. The chemical reaction formula is as follows: Figure 3 shown.

[0137] The synthesis method of indole-1-(4-fluorobenzyl)-3-carboxylic acid methyl ester is as follows: in an ice water bath, 2.5g of indole-3-carboxylic acid methyl ester, 1.75g ​​of potassium tert-butoxide and 100mL of tetrahydrofuran are placed in a three-necked flask, stirred for 1 hour, and then 2.82g of p-fluorobenzyl bromide is added, and stirred at room temperature overnight. After the reaction is complete, pour into 100mL of water, extract with ethyl acetate for several times, combine the organic phases, dry with anhydrous magnesium sulfate, and evaporate the solvent to obtain colorless liquid indole-1-(4-fluorobenzyl)-3-carboxylic acid methyl ester. The chemical reaction formula is as follows Figure 4 shown.

[0138] The synthesis method of indole-3-carboxylic acid methyl ester is as follows: in an ice water bath, 9.0 g of indole-3-carboxylic acid and 150 mL of methanol are placed in a three-necked flask, and 9 mL of concentrated sulfuric acid is added dropwise. After the addition is complete, heat to vortex and react for 24 hours. After the reaction is complete, evaporate the solvent under reduced pressure, add ethyl acetate and water, extract several times, dry the organic phase with anhydrous magnesium sulfate, evaporate the solvent, a white solid precipitates, filter, and obtain white solid indole-3-carboxylic acid methyl ester. The chemical reaction formula is as follows Figure 5 shown.

[0139] The drugs described in this application were all purchased from Sigma-Aldrich.

[0140] Example 2

[0141] Example 2 provides a method for using a synthetic cannabinoid detection kit. The specific implementation method is the same as Example 1, except that the synthetic cannabinoid complete antigen 2# is 5F-ADB complete antigen 2#.

[0142] The synthetic cannabinoid complete antigen 1# is 5F-ADB complete antigen 1#.

[0143] The hapten is 5F-ADB hapten.

[0144] The synthesis method of the 5F-ADB hapten is as follows: 0.55g 5F-ADB is dissolved in 20mL methanol, 2.2mL 1mol / L sodium hydroxide solution is added dropwise, and stirred at room temperature overnight. After the reaction is complete, the solvent is evaporated under reduced pressure, water is added, the pH is adjusted to 3-4 with HCl, and extracted with ether for multiple times, the organic phases are combined, dried with anhydrous magnesium sulfate, and the solvent is evaporated to obtain a white solid. The chemical reaction formula is as follows: Figure 6 shown.

[0145] The synthesis method of 5F-ADB is as follows: 0.5 g of indole-1-(5-fluoropentyl)-3-carboxylic acid, 1.09 g of Py-Bop and 10 mL of DMSO are placed in a three-necked flask, 1.1 g of L-tert-leucine methyl ester is added dropwise while stirring, and stirred at room temperature overnight. After the reaction is complete, it is poured into a sodium bicarbonate solution, extracted with ether, and purified by column chromatography to obtain a crystalline solid. The chemical reaction formula is as follows: Figure 7 shown.

[0146] The synthesis method of indole-1-(5-fluoropentyl)-3-carboxylic acid is as follows: 1.4 g of indole-1-(5-fluoropentyl)-3-carboxylic acid methyl ester is dissolved in 30 mL of methanol, 8 mL of 1 mol / L sodium hydroxide solution is added dropwise, and stirred at room temperature overnight. After the reaction is complete, the solvent is evaporated under reduced pressure, the white solid is dissolved in water, the pH is adjusted to 3-4 with HCl, and extracted with ethyl acetate for multiple times, the organic phases are combined, dried with anhydrous magnesium sulfate, and the solvent is evaporated to obtain a white solid indole-1-(5-fluoropentyl)-3-carboxylic acid. The chemical reaction formula is as follows Figure 8 shown.

[0147] The synthesis method of indole-1-(5-fluoropentyl)-3-carboxylic acid methyl ester is as follows: in an ice water bath, 2.0g indole-3-carboxylic acid methyl ester, 1.4g potassium tert-butoxide and 80mL tetrahydrofuran are placed in a three-necked flask, stirred for 1 hour, and then 2.02g 1-bromo-5-fluoropentane is added, and stirred at room temperature overnight. After the reaction is complete, pour into 100mL water, extract with ethyl acetate for several times, combine the organic phases, dry with anhydrous magnesium sulfate, and evaporate the solvent to obtain colorless liquid indole-1-(5-fluoropentyl)-3-carboxylic acid methyl ester. The chemical reaction formula is as follows Fig. 9 shown.

[0148] Comparative Example 1

[0149] Comparative Example 1 provides a detection kit for synthetic cannabinoids, and the specific implementation method is the same as that of Example 1, except that: the carrier protein 2# is BSA.

[0150] Comparative Example 2

[0151] Comparative Example 2 provides a detection kit for synthetic cannabinoids, and the specific implementation method is the same as that of Example 1, except that: the coupling agent is N-hydroxysuccinimide (NHS).

[0152] Comparative Example 3

[0153] Comparative Example 3 provides a detection kit for synthetic cannabinoids, and the specific implementation method is the same as that of Example 1, except that the weight ratio of the hapten to the carrier protein is 1:1.

[0154] Comparison Example 4

[0155] Comparative Example 4 provides a detection kit for synthetic cannabinoids, and the specific implementation method is the same as that of Example 2, except that: the carrier protein 2# is BSA.

[0156] Performance Testing Methods

[0157] 1. Sensitivity test results

[0158] For the synthetic cannabinoid kits prepared in Example 1 and Comparative Examples 1-3, ADB FUBINACA and AMB FUBINACA were respectively prepared into the following concentrations using a 0.01 M PBS diluent at pH 7.4, and the results were tested and observed. The results of ADB FUBINACA and AMB FUBINACA are respectively recorded in Table 1.

[0159] For the synthetic cannabinoid kits prepared in Example 2 and Comparative Example 4, 5F-ADB was diluted with 0.01 M PBS at pH 7.4 to prepare the following concentrations, and the results were detected and observed. The results are recorded in Table 2.

[0160] 2. Specificity test results

[0161] For the synthetic cannabinoid kits prepared in Examples 1-2 and Comparative Examples 1-4, 100 μg / ml of morphine, methamphetamine, ephedrine, pseudoephedrine, phenobarbital, cocaine, ketamine, methadone, tramadol, ranitidine, gatifloxacin, and procaine were prepared, and the concentration of Δ 9 -Tetrahydrocannabinolic acid samples were used as test samples, and the results were tested and observed, and the results were recorded in Table 3.

[0162] 3. Accelerated stability test

[0163] After the components of the kit in Example 1 were placed in a 37°C incubator for 7 days, their sensitivity to various concentrations of AMB FUBINACA was measured, and their color changes were observed. The colors were G10-G1 from dark to light, and compared with those at room temperature. The results are shown in Table 4.

[0164] The results showed that the kit underwent an accelerated stability test and its sensitivity and color remained unchanged after 7 days at 37°C, indicating that the kit has good stability and can be stored stably if the components are stored in accordance with the instructions.

[0165] Table 1 ADB FUBINACA / AMB FUBINACA test results

[0166]

[0167]

[0168] Table 2 5F-ADB test results

[0169] Concentration (ng / mL) Example 2 Comparative Example 2 0 - / - - / - 100 - / - - / - 250 - / - - / - 500 + / + - / - 1000 + / + - / - 1500 + / + - / -

[0170] Table 3 Specificity test results

[0171]

[0172]

[0173] Table 4 Accelerated stability test

[0174]

Claims

1. A detection kit for synthetic cannabinoids, characterized in that: It comprises a reagent strip and a plastic part; the reagent strip comprises a backing plate, a sample pad, a colloidal gold binding pad, a coating film and a water-absorbing pad; The colloidal gold binding pad is prepared by: adsorbing a synthetic cannabinoid colloidal gold labeled antibody onto glass fiber and drying the resulting mixture; The raw materials for preparing the synthetic cannabinoid colloidal gold labeled antibody include gold label diluent and synthetic cannabinoid antibody gold label solution; The preparation method of the synthetic cannabinoid antibody gold-labeled solution is as follows: a colloidal gold solution is mixed with a synthetic cannabinoid antibody, and the mixture is centrifuged to obtain the solution; the concentration of the synthetic cannabinoid antibody in the colloidal gold solution is 0.8-1.4 mg / 100 mL; The synthetic cannabinoid antibody is a polyclonal antibody or a monoclonal antibody; the polyclonal antibody is AMB-FUBINACA rabbit anti-polyclonal antibody and 5F-ADB rabbit anti-polyclonal antibody; the monoclonal antibody is AMB-FUBINACA mouse anti-monoclonal antibody and 5F-ADB mouse anti-monoclonal antibody; The preparation method of the polyclonal antibody is as follows: immunizing New Zealand white rabbits with synthetic cannabinoid complete antigen 2#, with an immunization dose of 0.8-1.2 mg / time, and injecting at multiple points subcutaneously on the back; The synthetic cannabinoid complete antigen 2# is AMB-FUBINACA complete antigen 2# or 5F-ADB complete antigen 2#; The preparation method of the synthetic cannabinoid complete antigen 2# is as follows: dissolving the hapten in an organic solvent, adding a coupling agent, and stirring for 2-6 hours to obtain a solution A; dissolving the carrier protein 2# in a buffer solution, dripping the solution A while stirring, and continuing to stir for 10-14 hours after the dripping is completed, and finally dialyzing in a dialysate at low temperature for three days to obtain the solution; The carrier protein 2# is hemocyanin; The hapten is AMB-FUBINACA hapten or 5F-ADB hapten; The structural formula of the AMB-FUBINACA hapten is Formula 1; the structural formula of the 5F-ADB hapten is Formula 2; Formula 1 Formula 2.

2. An application of the detection kit for synthetic cannabinoids according to claim 1, characterized in that: Applied to the detection of synthetic cannabinoid drugs Natasha.

3. A method for detecting synthetic cannabinoids, characterized in that: Add 3 to 4 drops of the test sample to the test kit for synthetic cannabinoids according to any one of claims 1 to 2, and then observe and determine the test result.

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