An Enzyme-Linked Immunosorbent Assay Kit for Detecting Cannabidiol and Its Application

Abstract

An enzyme-linked immunosorbent assay kit for detecting cannabidiol and its application. The kit includes: an enzyme-labeled plate coated with a coating antigen, a cannabidiol standard solution, a cannabidiol-specific antibody, an enzyme-labeled secondary antibody, a chromogenic solution, a termination solution, a washing solution, and a reconstitution solution. The coating antigen is a cannabidiol-conjugated antigen, and the cannabidiol-conjugated antigen is a conjugate of a cannabidiol hapten and a carrier protein. The cannabidiol hapten prepared in the present invention not only retains the characteristic structure of cannabidiol to the greatest extent, but also has a linker with a suitable structure for conjugation with the carrier protein, allowing the characteristic structure of cannabidiol to be fully exposed to the body as much as possible, enhancing the immune effect, improving the specificity of the antibody, and reducing the cross-reaction with other cannabidiol analogs. The enzyme-linked immunosorbent assay kit of the present invention can be used to detect the content of cannabidiol in industrial hemp samples, is easy to operate, low in cost, high in sensitivity, capable of on-site monitoring, and suitable for screening a large number of samples.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme-linked immunosorbent assay, and particularly relates to an enzyme-linked immunosorbent assay kit for cannabidiol and its application, which can qualitatively and quantitatively detect the content of cannabidiol in industrial hemp. Background Art

[0002] Cannabidiol, namely CBD, is a phytocannabinoid extracted from cannabis, lacking psychoactivity and having analgesic, anti-inflammatory, anti-tumor and chemopreventive activities. Cannabidiol can block the adverse effects of certain polyphenols on the human nervous system, and has a series of physiological active functions such as blocking breast cancer metastasis, treating epilepsy, anti-rheumatoid arthritis, anti-insomnia, etc., and has a good effect on treating multiple sclerosis.

[0003] Industrial hemp refers to hemp with a tetrahydrocannabinol (THC) content of less than 0.3%, which is an annual herbaceous plant of the genus Cannabis in the family Cannabaceae. The extract of cannabidiol (CBD) from the flowers and leaves of industrial hemp has broad market prospects. The determination of the content of cannabidiol in industrial hemp is helpful for monitoring the CBD content during the planting process of industrial hemp and optimizing industrial hemp varieties.

[0004] Currently, the commonly used methods for detecting cannabidiol are mainly instrumental methods, such as high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. Using these analytical methods not only requires expensive instruments and professional technicians, but also has a complex sample pretreatment process, a long cycle and high costs, making it difficult to meet the needs of rapid detection of a large number of samples and on-site samples. The immunoassay method based on the specific recognition of antigen and antibody can qualitatively and quantitatively detect the content of cannabidiol in samples.

[0005] The enzyme-linked immunosorbent assay kit is a highly efficient, sensitive and rapid sample detection method, and is the most widely used technology in enzyme immunoassay technology. However, there is no relevant report on the enzyme-linked immunosorbent assay kit for detecting cannabidiol in the current prior art. Summary of the Invention

[0006] The object of the present invention is precisely based on the above-mentioned current technical situation to provide an enzyme-linked immunosorbent assay kit capable of detecting the content of cannabidiol in industrial hemp, and to provide a rapid, simple and efficient qualitative and quantitative detection method applicable to a large number of samples.

[0007] A cannabidiol enzyme-linked immunosorbent assay kit, comprising an enzyme-labeled plate coated with a coating antigen, a cannabidiol standard solution, a cannabidiol-specific antibody, an enzyme-labeled secondary antibody, a chromogenic solution, a termination solution, a washing solution, and a reconstitution solution. The coating antigen is a cannabidiol-conjugated antigen. The enzyme-labeled secondary antibody is an enzyme-labeled goat anti-mouse secondary antibody. The cannabidiol-conjugated antigen is a conjugate of a cannabidiol hapten and a carrier protein. The cannabidiol hapten is prepared by the following method: reacting 3,5-dimethoxybenzaldehyde with triethyl-4-phosphide to form ethyl (2E,4E)-5-(3,5-dimethoxyphenyl)-2,4-pentadienoate, followed by reduction with PdC / H2, demethylation with HBr / HoAc, and saponification to obtain 5-(3,5-dihydroxyphenyl)pentanoic acid, and finally reacting with (1S,4R)-1-methyl-4-(1-methylethenyl)-2-cyclohexen-1-ol. The specific reaction process is as follows:

[0008]

[0009] The carrier protein is hemocyanin, bovine serum albumin, ovalbumin, human serum albumin, or thyroprotein.

[0010] The cannabidiol-specific antibody is prepared using the cannabidiol-conjugated antigen as an immunogen. The cannabidiol-specific antibody can be a cannabidiol monoclonal antibody or a cannabidiol polyclonal antibody, preferably a cannabidiol monoclonal antibody.

[0011] The labeling enzyme of the enzyme-labeled secondary antibody is horseradish peroxidase or bacterially extracted alkaline phosphatase, with horseradish peroxidase being preferred; the enzyme-labeled secondary antibody is obtained by conjugating an enzyme and a goat anti-mouse secondary antibody.

[0012] The cannabidiol standard solution includes 7 bottles with concentrations of 0, 0.01 ug / mL, 0.05 ug / mL, 0.25 ug / mL, 1 ug / mL, 5 ug / mL, and 20 ug / mL, respectively.

[0013] When the labeling enzyme is horseradish peroxidase, the chromogenic solution is a single-component TMB chromogenic solution or a two-component TMB chromogenic solution, and the termination solution is 1 - 2 mol / L sulfuric acid or 2 - 4 mol / L hydrochloric acid. When the labeling enzyme is bacterially extracted alkaline phosphatase, the substrate chromogenic solution is a p-nitrophenyl phosphate buffer solution, and the termination solution is 1 - 2 mol / L sodium hydroxide solution.

[0014] The washing solution is preferably a phosphate buffer solution with a pH value of 7.4, containing 0.01% - 0.5% Tween-20, 0.01% - 0.2% Proclin300 preservative, and 0.01 - 0.1 mol / L.

[0015] Among them, the coating buffer used in the preparation process of the enzyme-linked immunosorbent assay (ELISA) plate is a carbonate buffer with a pH value of 9.6 and a concentration of 0.05 mol / L, and the blocking solution is a phosphate buffer with a pH value of 7.1 - 7.5, containing 3% - 10% skim milk powder and 0.01 - 0.1 mol / L.

[0016] All percentages mentioned in the present invention are mass percentages.

[0017] The preparation process of the ELISA plate in the present invention is as follows: Dilute the coating antigen with the coating buffer to 0.1 μg / mL, add 100 μL to each well, incubate overnight at 4°C, discard the liquid in the wells by centrifugation, wash the wells 3 times with the washing solution, pat dry, then add 100 - 200 μL of the blocking solution to each well, incubate at 37°C in the dark for 2 h, discard the liquid in the wells by centrifugation, wash the wells 3 times with the washing solution, pat dry, and store in a vacuum-sealed aluminum foil bag after drying.

[0018] The present invention also provides a method for detecting cannabidiol using the above-mentioned ELISA kit for cannabidiol, which includes the following steps:

[0019] 1) Pretreat the sample to be tested to obtain a sample solution to be tested;

[0020] 2) Detect the sample solution to be tested using the above-mentioned ELISA kit for cannabidiol;

[0021] 3) Analyze the detection results.

[0022] The detection principle of the present invention is as follows:

[0023] This kit uses the competitive ELISA method. The conjugate antigen is pre-coated on the microtiter strips of the ELISA plate. The free cannabidiol in the sample and the conjugate antigen pre-coated on the microtiter strips of the ELISA plate compete for the anti-cannabidiol antibody. After adding the enzyme-labeled secondary antibody, TMB substrate is used for color development. The absorbance value of the sample is negatively correlated with the content of cannabidiol in it. By comparing with the standard curve and then multiplying by its corresponding dilution factor, the content of cannabidiol in the sample can be obtained.

[0024] The advantages of the present invention are as follows:

[0025] 1) The ELISA kit for detecting cannabidiol in the present invention mainly uses the enzyme-linked immunosorbent assay method to qualitatively or quantitatively detect the content of cannabidiol in the sample; the sample pretreatment process is simple and rapid, and a large number of samples can be detected simultaneously and quickly.

[0026] 2) The CBD hapten adopted in the present invention not only retains the characteristic structure of CBD to the greatest extent, but also has a connecting arm with a suitable structure when conjugated with the carrier protein, allowing the characteristic structure of CBD to be fully exposed to the body as much as possible, enhancing the immune effect, improving the specificity of the antibody, and reducing the cross-reaction with other CBD analogues; the CBD hapten prepared in the present invention introduces a carboxyl group into the original structure, which is beneficial to the preparation of the CBD artificial antigen; the CBD artificial antigen provided by the present invention has strong immunogenicity, which is beneficial to stimulating the body to complete the immune response, thereby obtaining high-quality monoclonal antibodies and providing a core reagent for the establishment of the CBD immunoassay method.

[0027] 3) The hapten adopted in the present invention has a high degree of overlap with the skeleton structure of the analyte CBD, effectively improving the immunogenicity of the CBD artificial antigen. The hapten has a connecting arm with a suitable structure when conjugated with the carrier protein, reducing the steric hindrance, allowing the characteristic structure of CBD to be fully exposed to the body as much as possible, enhancing the immune effect, and further improving the antibody affinity.

[0028] 4) The main reagents are provided in the form of working solutions, and the test method is simple and easy to perform, with characteristics such as high specificity, high sensitivity, and high accuracy. The ELISA kit of the present invention has a simple structure, is easy to operate, inexpensive, accurate, and is suitable for qualitative and quantitative detection of large batches of sample screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Synthetic route diagram of the CBD hapten;

[0030] Figure 2 : Standard curve graph of the kit;

[0031] Figure 3 : 1H NMR spectrum of the CBD hapten;

[0032] Figure 4 : 13C NMR spectrum of the CBD hapten;

[0033] Figure 5 : UV absorption spectrum of the CBD complete antigen. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, those skilled in the art may make various modifications or decorations to the present invention within the scope defined by the appended claims, and these modifications or decorations should also fall within the protection scope of the invention.

[0035] Example 1 Preparation of Kit Components

[0036] 1. Preparation of the CBD Hapten

[0037] Take 0.87 g (22 mmol) of sodium hydride, add 20 ml of dry and anhydrous tetrahydrofuran and stir to form a suspension. Dropwise add 50 ml of anhydrous tetrahydrofuran containing 5 g of triethyl-4-phosphide (20 mmol) dropwise under an ice bath, and stir the reaction for 30 min. Subsequently, dropwise add 50 ml of anhydrous tetrahydrofuran containing 2.77 g of 3,5-dimethoxybenzaldehyde, remove the ice bath, and stir the reaction at room temperature for 1 h, then heat to 60 °C and react for 1 h. After determining the end of the reaction by TLC, cool the reaction solution to room temperature, quench the reaction with 120 ml of water, extract the aqueous phase 3 times with 120 ml of ethyl acetate, combine the organic phases, back-extract the organic phase with saturated brine, dry over anhydrous sodium sulfate overnight, and rotary evaporate to obtain 4.0 g of intermediate 1.

[0038] Add 3.6 g of intermediate 1 and 10% Pd-C (0.74 g) to 75 ml of methanol, add hydrogen and stir the reaction at room temperature for 2 h, filter Pd-C, concentrate the filtrate to obtain 3.5 g of a pale yellow oily liquid (intermediate 2).

[0039] Add 50 ml of 40% hydrobromic acid and 50 ml of glacial acetic acid to intermediate 2, reflux the reaction at 60 °C for 4 h. After determining the end of the reaction by TLC, cool the reaction mixture to room temperature and add 150 ml of ice water. Extract the reaction mixture 3 times with 150 ml of ethyl acetate, back-extract the organic phase with saturated brine, dry over anhydrous sodium sulfate overnight, and concentrate the organic phase to obtain a black oily crude product. The crude product is purified by column chromatography (ethyl acetate / hexane 1:2) to obtain 2.50 g of intermediate 3.

[0040] Take 1 g of intermediate 3 (4.75 mmol) and 0.18 g of p-TsOH (0.95 mmol), add them to 50 ml of a THF:DCM (1:4) mixture, stir and mix well, dropwise add 1.08 g of (1S,4R)-1-methyl-4-(1-methylethenyl)-2-cyclohexen-1-ol, stir the reaction at room temperature for 30 min, determine the progress of the reaction by TLC, finally add 50 ml of ethyl acetate to terminate the reaction, and adjust the pH value of the solution to 3 - 4 with a NaHCO3 solution. Separate the organic phase, wash it twice with saturated brine, dry and concentrate to obtain 2.3 g of an oily crude product. After simple purification by column chromatography and further purification by preparative HPLC, 220 mg of the cannabidiol hapten is obtained.

[0041] For the cannabidiol hapten 1 1H NMR, 13 13C NMR is as Figure 3-4 shown.

[0042] 2. Preparation of antigen

[0043] Preparation of immunogen: The hapten of cannabidiol is conjugated with bovine serum albumin to obtain the immunogen.

[0044] Take 20 mg of hapten, 23 mg of EDC, and 14 mg of NHS, add them to 1 ml of dioxane and dissolve completely to obtain solution A, and stir and react at room temperature for 4 h. Take 50 mg of bovine serum albumin and dissolve it completely with 5 ml of 10 mM PBS by stirring to obtain solution B. Dropwise add solution A into solution B, and stir and react at 4 °C overnight. After the reaction, dialyze the protein solution with 10 mM PBS for 3 days, changing the solution every 6 h, to obtain the cannabidiol-bovine serum albumin conjugate, i.e., the immunogen CBD-BSA.

[0045] Preparation of coating antigen: The hapten of cannabidiol is conjugated with ovalbumin to obtain the coating antigen.

[0046] Take 10 mg of hapten, 12 mg of EDC, and 7 mg of NHS, add them to 1 ml of anhydrous DMF and dissolve completely to obtain solution A, and stir and react at room temperature for 4 h. Take 20 mg of ovalbumin and dissolve it completely with 5 ml of 10 mM PBS by stirring to obtain solution B. Dropwise add solution A into solution B, and stir and react at 4 °C overnight. After the reaction, dialyze the protein solution with 10 mM PBS for 3 days, changing the solution every 6 h, to obtain the cannabidiol-ovalbumin conjugate, i.e., the coating antigen CBD-OVA.

[0047] Identification of the complete antigen of cannabidiol

[0048] The complete antigen is identified for its conjugation result by ultraviolet spectroscopy, and the conjugation ratio is calculated using the concentrations of the small molecule and the protein in the conjugate. The maximum absorption peak of the cannabidiol hapten-carrier protein has changed significantly compared with the maximum absorption peaks of the cannabidiol hapten and the carrier protein, indicating that the preparation of the cannabidiol-carrier protein is successful (see Figure 5 ). After calculation, the conjugation ratio of the hapten to BSA is 25:1, and the conjugation ratio to OVA is 16:1.

[0049] 3. Preparation of monoclonal antibodies against cannabidiol

[0050] Animal immunization: Select 6-8-week-old female Balb / c mice, mix the immunogen with Freund's adjuvant for emulsification, and use an immunization dose of 100 μg / mouse to make the mice produce antiserum.

[0051] Cell fusion and subcloning: Take the spleen cells of the successfully immunized Balb / c mice, adjust the ratio of myeloma cells SP2 / 0 to the immunized mouse spleen cells to 1:5 - 1:10 for fusion, use the competitive ELISA method to measure the cell culture supernatant, and screen out the appropriate positive wells. Use the limiting dilution method to clone the positive wells until a hybridoma cell line that stably secretes monoclonal antibodies is obtained.

[0052] Preparation and purification of monoclonal antibody: Female Balb / c mice aged 10 - 11 weeks were selected. Adjuvant was injected into the peritoneal cavity of Balb / c mice, 0.3 - 0.5 ml per mouse, and the peritoneal cavity was treated for about 7 - 15 days. The selected hybridoma cell line was expanded in culture and then inoculated into the peritoneal cavity of Balb / c mice. The mice were sacrificed by cervical dislocation, and the ascites were extracted. The ascites were centrifuged at 3000 rpm for 10 min. The ascites were first purified by ammonium sulfate precipitation and then purified by Protein G column to obtain the final monoclonal antibody.

[0053] Determination of the titer of monoclonal antibody

[0054] The titer of the antibody determined by Elisa method was 1:600000.

[0055] Competitive Elisa: The enzyme - linked immunosorbent assay (ELISA) plate was coated with cannabidiol - ovalbumin (CBD - OVA). After adding cannabidiol standard and cannabidiol monoclonal antibody and incubating at 37 °C for 2 h, it was washed. Then, HRP - labeled goat anti - mouse secondary antibody was added. After washing again, TMB chromogenic solution was added, and the reaction was terminated with acid. The absorbance value at 450 nm was measured on an enzyme - linked immunosorbent assay reader.

[0056] Determination of the specificity of monoclonal antibody

[0057] The specificity of an antibody refers to its ability to recognize the corresponding antigen or similar antigenic substances. The higher the specificity of the antibody, the stronger its recognition ability. Specificity is usually measured by the cross - reaction rate. The cross - reaction rate can be determined by a competitive inhibition assay. Competitive inhibition curves were made with different concentrations of antigen and similar antigen respectively, the binding rate of each was calculated, the concentration at IC50 of each was determined, and the cross - reaction rate was calculated according to the following formula.

[0058] Cross - reaction rate =

[0059] Calculated according to the above formula, the cross - reaction rates of the antibody of the present invention with other main components of cannabis were tested: tetrahydrocannabinol (THC) < 1%, cannabinol (CBC) < 1%, cannabinol (CBN) < 1%, cannabigerol (CBG) < 1%. The antibody of the present invention had basically no cross - reaction with tetrahydrocannabinol (THC), cannabinol (CBC), cannabinol (CBN), and cannabigerol (CBG), indicating that the antibody had good specificity.

[0060] 4. Preparation of enzyme - labeled secondary antibody

[0061] It was obtained by coupling goat anti - mouse secondary antibody with horseradish peroxidase (HRP).

[0062] 5. Preparation of ELISA plate

[0063] Dilute the coated antigen with the coating buffer to 0.1 μg / mL, add 100 μL to each well, coat overnight at 4°C, drain the liquid, wash 3 times with 0.05% PBST, pat dry, then add 200 μL of 5% skim milk powder to each well to block the ELISA plate, block at 37°C for 2 h, drain the liquid, wash 3 times with 0.05% PBST, pat dry, and store in a vacuum-sealed aluminum foil bag after complete drying in an oven.

[0064] Example 2 Preparation of an Enzyme-Linked Immunosorbent Assay Kit for Detecting Cannabidiol

[0065] Prepare an enzyme-linked immunosorbent assay kit for detecting cannabidiol, using the following components:

[0066] (1) ELISA plate coated with cannabidiol-conjugated antigen;

[0067] (2) 7 bottles of cannabidiol standard solutions with concentrations of 0, 0.01 μg / mL, 0.05 μg / mL, 0.25 μg / mL, 1 μg / mL, 5 μg / mL, and 20 μg / mL respectively;

[0068] (3) Goat anti-mouse secondary antibody labeled with horseradish peroxidase;

[0069] (4) The chromogenic solution is a single-component TMB chromogenic solution;

[0070] (5) The stop solution is 2 mol / L sulfuric acid;

[0071] (6) The washing solution has a pH value of 7.4 and contains 0.05% Tween-20, 0.05% sodium azide preservative, and 0.01 mol / L phosphate buffer solution;

[0072] (7) The reconstitution solution is a phosphate buffer solution with a pH value of 7.4 and a concentration of 0.01 mol / L.

[0073] Example 3 Detection of Cannabidiol Content in Industrial Hemp

[0074] 1. Sample Pretreatment

[0075] Weigh 10 mg of industrial hemp sample into a 5 mL grinding tube, add 1 mL of methanol, grind for four minutes, let it stand, take 50 μL of the supernatant and add it to 950 μL of the reconstitution solution, mix well, and take 50 μL for analysis.

[0076] 2. Kit Detection

[0077] Add 50 μL of the standard / sample to the corresponding microplate wells of the microplate reader, then add 50 μL / well of the antibody working solution, mix well by shaking, incubate at 37 °C for 1 h, discard the liquid by centrifugation, wash 3 times with 200 μL / well of the washing solution, and discard the liquid by centrifugation. Add 100 μL / well of the enzyme-labeled secondary antibody, mix well by shaking, incubate at 37 °C for 0.5 h, take out and repeat the plate washing. Add 100 μL / well of the chromogenic solution, incubate at 37 °C for 15 min, and mix well by shaking. Add 50 μL / well of the stop solution. Set the microplate reader at 450 nm with a reference wavelength of 620 nm, and measure the OD value of each well.

[0078] 3. Analysis of the test results

[0079] The percentage absorbance of the cannabidiol standard or sample is equal to the absorbance value of the standard or sample divided by the average absorbance value of the first standard, and then multiplied by 100% to obtain the percentage absorbance value of the standard or sample. Using the percentage absorbance of the standard as the ordinate and the logarithm of the cannabidiol standard concentration as the abscissa, plot the standard curve. Substitute the percentage absorbance of the sample into the standard curve, read the corresponding concentration of the sample from the standard curve, and multiply by its corresponding dilution factor to obtain the actual concentration of cannabidiol in the sample.

[0080] Example 4 Technical parameters of the cannabidiol kit

[0081] 1. Sensitivity and detection limit

[0082] In ELISA, it can be determined by measuring the optical density value of the "0" standard tube. Measure 10 or more "0" standard tubes, calculate the average value X of the optical density values, and then subtract twice the standard deviation (SD). Check the concentration corresponding to the optical density value of X - 2SD from the standard curve, which is the sensitivity.

[0083] The detection limit is for the "sample" and usually refers to the minimum detectable amount of the actual sample measured by the kit product. Its theoretical definition is: measure 20 negative (blank) samples according to a reasonable pretreatment method, calculate the average value X and the standard deviation (SD), and the result obtained according to the formula X + 3SD is the detection (lower) limit of the sample. Its value is closely related to the sample type, sampling area, and sample pretreatment method.

[0084] Measuring the sensitivity of the kit according to the conventional method, the range of the standard curve is 0 - 20 μg / mL, IC50 = 0.2 μg / mL, and its detection limit for industrial hemp samples is 0.05 μg / mL (CBD content 0.01%).

[0085] 2. Comparison between the detection of cannabidiol content in industrial hemp samples by the kit and chromatographic results

[0086] Sample number Chromatographic result Kit result Recovery rate 1 0.22% 0.24% 109% 2 1.34% 1.37% 102% 3 2.04% 2.25% 110% 4 0.82% 0.87% 106% 5 0.38% 0.37% 97%

[0087] 3. Kit Stability

[0088] The storage condition of the kit is 2 - 8°C. After 12 months of determination, the maximum absorbance value (zero standard), 50% inhibition concentration, and the measured value of the cannabidiol standard addition sample of the kit are all within the normal range. Considering that there may be abnormal storage conditions during transportation and use, the kit was placed at 37°C for 7 days for an accelerated aging experiment. The results show that all indicators of the kit fully meet the requirements. From the above results, it can be concluded that the kit can be stored at 2 - 8°C for at least 12 months or more.

Claims

1. A cannabidiol enzyme-linked immunosorbent assay kit, characterized in that: It includes an enzyme-labeled plate coated with a coating antigen, a cannabidiol standard solution, a cannabidiol-specific antibody, an enzyme-labeled secondary antibody, a chromogenic solution, a termination solution, a washing solution, and a reconstitution solution. The coating antigen is a cannabidiol-conjugated antigen. The enzyme-labeled secondary antibody is an enzyme-labeled goat anti-mouse secondary antibody. The cannabidiol-conjugated antigen is a conjugate of a cannabidiol hapten and a carrier protein. The cannabidiol hapten is prepared by the following method: reacting 3,5-dimethoxybenzaldehyde with triethyl-4-phosphide to form ethyl (2E,4E)-5-(3,5-dimethoxyphenyl)-2,4-pentadienoate, followed by reduction with PdC / H2, demethylation with HBr / HoAc, and saponification to obtain 5-(3,5-dihydroxyphenyl)pentanoic acid, and finally reacting with (1S,4R)-1-methyl-4-(1-methylethenyl)-2-cyclohexen-1-ol. The structural formula of the cannabidiol hapten is as follows: The specific reaction process is as follows: .

2. The cannabidiol enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The specific method and reaction process are as follows: 1) Take 0.87 g of 22 mmol sodium hydride, add 20 ml of dry and anhydrous tetrahydrofuran and stir to form a suspension. Dropwise add 50 ml of anhydrous tetrahydrofuran containing 5 g of triethyl-4-phosphide dropwise under an ice bath, stir and react for 30 min. Then dropwise add 50 ml of anhydrous tetrahydrofuran containing 2.77 g of 3,5-dimethoxybenzaldehyde, remove the ice bath, and stir and react at room temperature for 1 h. Then heat to 60 °C and react for 1 h; after determining the end of the reaction by TLC, cool the reaction solution to room temperature, quench the reaction with 120 ml of water, extract the aqueous phase 3 times with 120 ml of ethyl acetate, combine the organic phases, back-extract the organic phase with saturated brine, dry over anhydrous sodium sulfate overnight, and rotary evaporate to obtain 4.0 g of intermediate 1; 2) Add 3.6 g of intermediate 1 and 0.74 g of 10% Pd-C to 75 ml of methanol, add hydrogen and stir and react at room temperature for 2 h, filter Pd-C, concentrate the filtrate to obtain 3.5 g of a pale yellow oily liquid, namely intermediate 2; 3) Add 50 ml of 40% hydrobromic acid and 50 ml of glacial acetic acid to intermediate 2, reflux and react at 60 °C for 4 h; after determining the end of the reaction by TLC, cool the reaction mixture to room temperature and add 150 ml of ice water; extract the reaction mixture 3 times with 150 ml of ethyl acetate, back-extract the organic phase with saturated brine, dry over anhydrous sodium sulfate overnight, and concentrate the organic phase to obtain a black oily crude product; purify the crude product by column chromatography to obtain 2.50 g of intermediate 3; 4) Take 1 g of intermediate 3 with a concentration of 4.75 mmol and 0.18 g of p-TsOH with a concentration of 0.95 mmol and add them to 50 ml of a mixed solution of THF:DCM = 1:4, stir and mix evenly, dropwise add 1.08 g of (1S,4R)-1-methyl-4-(1-methylethenyl)-2-cyclohexen-1-ol, stir and react at room temperature for 30 min, determine the progress of the reaction by TLC, finally add 50 ml of ethyl acetate to terminate the reaction, and adjust the pH value of the solution to 3-4 with a NaHCO3 solution, separate the organic phase, wash it twice with saturated brine, dry and concentrate to obtain 2.3 g of an oily crude product, and further purify it by preparative HPLC after simple purification by column chromatography to obtain 220 mg of cannabidiol hapten.

3. The cannabidiol enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The carrier protein is hemocyanin, bovine serum albumin, ovalbumin, human serum albumin or thyroprotein.

4. The cannabidiol enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The cannabidiol-specific antibody is a cannabidiol monoclonal antibody.

5. The cannabidiol enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The cannabidiol standard solution includes 7 bottles with concentrations of 0, 0.01 μg / mL, 0.05 μg / mL, 0.25 μg / mL, 1 μg / mL, 5 μg / mL, and 20 μg / mL respectively.

6. The cannabidiol enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: When the labeled enzyme is horseradish peroxidase, the chromogenic solution is a single-component TMB chromogenic solution or a two-component TMB chromogenic solution, and the termination solution is 1-2 mol / L sulfuric acid or 2-4 mol / L hydrochloric acid.

7. The cannabidiol enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: When the labeled enzyme is alkaline phosphatase extracted from bacteria, the substrate chromogenic solution is p-nitrophenyl phosphate buffer solution, and the termination solution is 1-2 mol / L sodium hydroxide solution.

8. The cannabidiol enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The pH value of the washing solution is 7.4, and it contains 0.01%-0.5% Tween-20, 0.01%-0.2% Proclin300 preservative, and 0.01-0.1 mol / L phosphate buffer solution.

9. A method for detecting the content of cannabidiol in a sample by using the cannabidiol enzyme-linked immunosorbent assay kit according to any one of claims 1-8, characterized in that: comprises the following steps: Performing pretreatment on the sample to be tested to obtain a sample solution to be tested; Detecting the sample solution to be tested by using the cannabidiol enzyme-linked immunosorbent assay kit according to any one of claims 1-8; Analyzing the detection result.

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