A test strip for detecting cannabidiol, its preparation method and application

Abstract

A test strip for detecting cannabidiol, its preparation method and application. The test strip includes a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate. The conjugate release pad is sprayed with a colloidal gold-labeled cannabidiol-specific antibody. The reaction membrane is fixed with a test line and a quality control line. The test line is coated with a cannabidiol hapten-carrier protein conjugate, and the quality control line is coated with goat anti-mouse secondary antibody. The cannabidiol-specific antibody is obtained using a cannabidiol-hapten carrier protein conjugate as an immunogen, and the cannabidiol hapten retains the characteristic structure of cannabidiol to the greatest extent. The present invention uses colloidal gold-labeled antibody and immunochromatography technology to achieve rapid immunoassay of cannabidiol in samples. The present invention also provides a method for detecting cannabidiol in samples using the above test strip, which has high sensitivity, rapid detection, convenient operation, is economical and practical, and can achieve rapid detection and on-site detection of a large number of samples.

Description

Technical Field

[0001] The present invention relates to the preparation and application of test strips, and particularly to a test strip for detecting cannabidiol, a preparation method thereof, and an application thereof, which can qualitatively detect 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 cannabis with a tetrahydrocannabinol (THC) content of less than 0.3%, which is an annual herb 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 cannabidiol content in industrial hemp is helpful for monitoring the CBD content during the cultivation 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. Immunoassay methods based on the specific recognition of antigen and antibody can qualitatively and quantitatively detect the cannabidiol content in samples.

[0005] The present invention applies the colloidal gold immunochromatography method to determine the content of cannabidiol in industrial hemp, which has the advantages of low detection limit, strong specificity, simple operation, fast detection speed, low detection cost, and is very easy to promote. At present, there is no relevant report on the colloidal gold test strip for detecting cannabidiol in the existing technology. Summary of the Invention

[0006] The purpose of the present invention is to provide a test strip capable of detecting cannabidiol in industrial hemp, a preparation method thereof, and to provide a rapid, simple and efficient qualitative detection method suitable for a large number of samples.

[0007] The purpose of the present invention is achieved by the following technical solutions:

[0008] A test strip for detecting cannabidiol, comprising a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate; wherein: a colloidal gold-labeled cannabidiol-specific antibody is sprayed on the conjugate release pad, and a test line and a quality control line are fixed on the reaction membrane. The test line is coated with a cannabidiol hapten-carrier protein conjugate, and the quality control line is coated with goat anti-mouse secondary antibody; the cannabidiol-specific antibody is obtained by using a cannabidiol hapten-carrier protein conjugate as an immunogen. The cannabidiol hapten is prepared by the following method: Ethyl (2E,4E)-5-(3,5-dimethoxyphenyl)penta-2,4-dienoate is generated by reacting 3,5-dimethoxybenzaldehyde with triethyl-4-phosphide, and then reduced by PdC / H2, demethylated by HBr / HoAc and saponified to obtain 5-(3,5-dihydroxyphenyl)pentanoic acid, and finally reacted with (1S,4R)-1-methyl-4-(1-methylethenyl)-2-cyclohexen-1-ol. See the specific reaction process in Figure 3

[0009] The cannabidiol conjugate antigen is obtained by conjugating a cannabidiol hapten with a carrier protein, and the carrier protein is hemocyanin, bovine serum albumin, ovalbumin, human serum albumin or thyroprotein.

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

[0011] The goat anti-mouse antibody is obtained by immunizing a sheep with a mouse-derived antibody.

[0012] The sample absorption pad, the conjugate release pad, the reaction membrane and the water absorption pad are sequentially pasted on the bottom plate, and 1 / 3 to 1 / 2 of the conjugate release pad is covered under the sample absorption pad.

[0013] The bottom plate can be a PVC bottom plate or other hard non-water-absorbing materials; the sample absorption pad can be filter paper or filter oil paper; the conjugate release pad can be glass fiber or polyester fiber; the water absorption pad is blotting paper; the reaction membrane can be a nitrocellulose membrane or an acetate cellulose membrane.

[0014] Another object of the present invention is to provide a method for preparing the above test strip, which comprises the steps of:

[0015] 1) Prepare a conjugate release pad sprayed with a colloidal gold-labeled cannabidiol-specific antibody;

[0016] 2) Prepare a reaction membrane with a test line coated with a cannabidiol hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse secondary antibody.

[0017] 3) Assemble the conjugate release pad, reaction membrane prepared in 1) and 2) with the sample absorption pad, water absorption pad and base plate into a test strip.

[0018] The specific steps include:

[0019] 1) Preparation of hapten: 3,5-dimethoxybenzaldehyde reacts with triethyl-4-phosphide to generate ethyl (2E,4E)-5-(3,5-dimethoxyphenyl)-2,4-pentadienoate, which is then reduced by PdC / H2, demethylated by HBr / HoAc and saponified to obtain 5-(3,5-dihydroxyphenyl)valeric acid, and finally reacts with (1S,4R)-1-methyl-4-(1-methylethenyl)-2-cyclohexen-1-ol to obtain the cannabidiol hapten;

[0020] 2) Couple the cannabidiol hapten with carrier protein to obtain the cannabidiol hapten-carrier protein conjugate;

[0021] 3) Immunize mice with the cannabidiol hapten-carrier protein conjugate, and fuse and screen the mouse spleen cells and SP2 / 0 myeloma cells to obtain the cannabidiol hybridoma cell line;

[0022] 4) Extract mouse IgG to immunize healthy goats to obtain goat anti-mouse secondary antibody;

[0023] 5) Prepare colloidal gold by reacting chloroauric acid with trisodium citrate;

[0024] 6) Add the cannabidiol monoclonal antibody prepared in step 3) to the colloidal gold prepared in step 5) to obtain the colloidal gold-labeled cannabidiol monoclonal antibody;

[0025] 7) Spray the colloidal gold-labeled cannabidiol monoclonal antibody on the conjugate release pad, take it out after drying at 37°C for 12 h, and store it in a dry environment for later use;

[0026] 8) Coat the reaction membrane with the cannabidiol hapten-carrier protein conjugate to form a test line, and coat the reaction membrane with goat anti-mouse secondary antibody to form a quality control line;

[0027] 9) Immerse the sample absorption pad in a phosphate buffer solution containing 0.2% Tween 20, 0.5% PVP K30, 0.5% bovine serum albumin, pH 7.4, and 0.02 mol / L for 2 h, dry it at 37°C for 24 h, and store it in a dry environment for later use;

[0028] 10) Paste the sample absorption pad, conjugate release pad, reaction membrane, and water absorption pad on the base plate in sequence. The sample absorption pad covers the conjugate release pad. Finally, cut it into small strips 4 mm wide, put in a desiccant, seal it in an aluminum foil bag, and it can be stored for 12 months at 4 - 30°C.

[0029] Another object of the present invention is to provide a method for detecting cannabidiol in industrial hemp using the above test strip, which comprises the steps of:

[0030] (1) Pretreating the sample to be tested to obtain a sample solution to be tested;

[0031] (2) Detecting with the test strip;

[0032] (3) Analyzing the test results.

[0033] The rapid detection test strip for cannabidiol of the present invention adopts a highly specific antibody-antigen reaction and immunochromatographic analysis technology. The cannabidiol antibody labeled with colloidal gold is fixed on the conjugate release pad. During the flow of the sample, the cannabidiol in the sample binds to the colloidal gold-labeled cannabidiol monoclonal antibody on the conjugate release pad to form a cannabidiol-antibody-colloidal gold label. The cannabidiol in the sample competes with the cannabidiol hapten-carrier protein conjugate on the test line of the reaction membrane for binding to the colloidal gold-labeled cannabidiol monoclonal antibody. Whether the content of cannabidiol in the sample solution to be tested exceeds the set threshold is judged according to the presence or absence or the color depth of the red strip on the test line.

[0034] The judgment of the test results is as Figure 2 shown.

[0035] Negative: The color development of the test line (T) is darker or the same as that of the quality control line (C), indicating that the content concentration of cannabidiol in the sample is lower than the detection limit, and it is judged as negative.

[0036] Positive: The color development of the test line (T) is lighter than that of the quality control line (C) or the test line (T) does not develop color, indicating that the concentration of cannabidiol in the sample is equal to or higher than the detection limit, and it is judged as positive.

[0037] Invalid: When the quality control line (C) does not show a red strip, the test strip is judged as invalid regardless of whether the test line (T) shows a red strip or not.

[0038] The advantages of the present invention are as follows:

[0039] 1. It fills the blank that there is currently no test strip for detecting cannabidiol.

[0040] 2. The cannabidiol hapten adopted by the present invention not only retains the characteristic structure of cannabidiol to the greatest extent, but also has a connecting arm with a suitable structure when conjugated with the carrier protein, so that the characteristic structure of cannabidiol is 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;

[0041] 3. The hapten of cannabidiol prepared by the present invention introduces a carboxyl group into the original structure, which is beneficial to the preparation of the artificial antigen of cannabidiol; the artificial antigen of cannabidiol provided by the present invention has strong immunogenicity, which is beneficial to stimulating the body to complete the immune response, so as to obtain high-quality monoclonal antibodies and provide core reagents for the establishment of the immunoassay method of cannabidiol.

[0042] 4. The hapten adopted by the present invention has a high degree of overlap with the skeleton structure of the analyte cannabidiol, effectively improving the immunogenicity of the artificial antigen of cannabidiol. There is a connecting arm with a suitable structure in the coupling of the hapten and the carrier protein, reducing the steric hindrance and exposing the characteristic structure of cannabidiol to the body as much as possible, enhancing the immune effect and further improving the antibody affinity.

[0043] 5. The test strip of the present invention has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, not limited by detection equipment, simple storage and long shelf life. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 : Schematic structural diagram of a test strip for detecting cannabidiol of the present invention,

[0045] In the figure: 1. Sample absorption pad; 2. Conjugate release pad; 3. Reaction membrane; 4. Water absorption pad; 5. Bottom plate; 6. Detection line; 7. Quality control line.

[0046] Figure 2 : Schematic diagram of the test result of the test strip sample;

[0047] Figure 3 : Synthetic route diagram of the cannabidiol hapten;

[0048] Figure 4 : 1H NMR spectrum of the cannabidiol hapten;

[0049] Figure 5 : 13C NMR spectrum of the cannabidiol hapten;

[0050] Figure 6 : UV absorption spectrum of the complete antigen of cannabidiol. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0051] 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.

[0052] Example 1 Preparation of the cannabidiol test strip

[0053] The preparation method of the test strip mainly includes the following steps:

[0054] 1) Prepare a conjugate release pad sprayed with a colloidal gold-labeled cannabidiol-specific antibody;

[0055] 2) Prepare a reaction membrane with a detection line coated with a cannabidiol hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse secondary antibody.

[0056] 3) Assemble the conjugate release pad and reaction membrane prepared in 1) and 2) with a sample absorption pad, a water absorption pad and a bottom plate into a test strip.

[0057] The following is a detailed description:

[0058] 1. Preparation of cannabidiol hapten

[0059] Take 0.87 g (22 mmol) of sodium hydride, add 20 ml of dry 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 ice bath, and stir for 30 min. Then dropwise add 50 ml of anhydrous tetrahydrofuran containing 2.77 g of 3,5-dimethoxybenzaldehyde, remove the ice bath, stir at room temperature for 1 h, and 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.

[0060] Add 3.6 g of intermediate 1 and 10% Pd-C (0.74 g) to 75 ml of methanol, stir and react with hydrogen 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).

[0061] Add 50 ml of 40% hydrobromic acid and 50 ml of glacial acetic acid to intermediate 2, reflux 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.

[0062] 1 g of intermediate 3 (4.75 mmol) and 0.18 g of p-TsOH (0.95 mmol) were added to 50 ml of a THF:DCM (1:4) mixture, stirred and mixed evenly, and 1.08 g of (1S,4R)-1-methyl-4-(1-methylethenyl)-2-cyclohexen-1-ol was added dropwise. The reaction was stirred at room temperature for 30 min, and the progress of the reaction was determined by TLC. Finally, 50 ml of ethyl acetate was added to terminate the reaction, and the pH value of the solution was adjusted to 3-4 with a NaHCO3 solution. The organic phase was separated, washed twice with saturated brine, dried and concentrated to obtain 2.3 g of an oily crude product. After simple purification by column chromatography, it was further purified by preparative HPLC to obtain 220 mg of the CBD hapten.

[0063] Of the CBD hapten 1 1H NMR, 13 13C NMR is as Figure 4-5 shown.

[0064] 2. Preparation of antigen

[0065] Preparation of immunogen: The CBD hapten was conjugated with bovine serum albumin to obtain the immunogen.

[0066] 20 mg of the hapten, 23 mg of EDC, and 14 mg of NHS were added to 1 ml of dioxane and dissolved completely to obtain solution A, and the reaction was stirred at room temperature for 4 h. 50 mg of bovine serum albumin was dissolved completely with 5 ml of 10 mM PBS to obtain solution B. Solution A was added dropwise to solution B, and the reaction was stirred at 4 °C overnight. After the reaction, the protein solution was dialyzed with 10 mM PBS for 3 days, and the solution was changed every 6 h to obtain the CBD-bovine serum albumin conjugate, i.e., the immunogen CBD-BSA.

[0067] Preparation of coating antigen: The CBD hapten was conjugated with ovalbumin to obtain the coating antigen.

[0068] 10 mg of the hapten, 12 mg of EDC, and 7 mg of NHS were added to 1 ml of anhydrous DMF and dissolved completely to obtain solution A, and the reaction was stirred at room temperature for 4 h. 20 mg of ovalbumin was dissolved completely with 5 ml of 10 mM PBS to obtain solution B. Solution A was added dropwise to solution B, and the reaction was stirred at 4 °C overnight. After the reaction, the protein solution was dialyzed with 10 mM PBS for 3 days, and the solution was changed every 6 h to obtain the CBD-ovalbumin conjugate, i.e., the coating antigen CBD-OVA.

[0069] Identification of the CBD complete antigen

[0070] The complete antigen was identified for its conjugation results by ultraviolet spectroscopy, and the conjugation ratio was calculated using the concentrations of the small molecule and the protein in the conjugate. The maximum absorption peak of the cannabidiol hapten-carrier protein changed significantly compared with those of the cannabidiol hapten and the carrier protein, indicating the successful preparation of the cannabidiol-carrier protein (see Figure 6 ). After calculation, the conjugation ratio of the hapten to BSA was 25:1, and the conjugation ratio to OVA was 16:1.

[0071] 3. Preparation of Monoclonal Antibodies Against Cannabidiol

[0072] Animal Immunization: Female Balb / c mice at 6 - 8 weeks of age were selected. The immunizing antigen was mixed and emulsified with Freund's adjuvant, and the mice were immunized at a dose of 100 μg per mouse to produce antiserum.

[0073] Cell Fusion and Subcloning: Spleen cells from immunized Balb / c mice were taken, and the ratio of myeloma cells SP2 / 0 to immunized mouse spleen cells was adjusted to 1:5 - 1:10 for fusion. The cell culture supernatant was measured by competitive ELISA to screen for suitable positive wells. The positive wells were cloned by the limiting dilution method until a hybridoma cell line that stably secreted monoclonal antibodies was obtained.

[0074] Preparation and Purification of Monoclonal Antibodies: Female Balb / c mice at 10 - 11 weeks of age 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 screened 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 and centrifuged at 3000 rpm for 10 min. The ascites were first purified by ammonium sulfate precipitation and then purified using a Protein G column to obtain the final monoclonal antibody.

[0075] Determination of the Titer of Monoclonal Antibodies

[0076] The titer of the antibody determined by the ELISA method was 1:600000.

[0077] Competitive ELISA: The ELISA plate was coated with cannabidiol-OVA. After adding the cannabidiol standard and the monoclonal antibody against cannabidiol and incubating at 37°C for 2 h, it was washed. Then, HRP-labeled goat anti-mouse secondary antibody was added, and after washing again, TMB chromogenic solution was added. The reaction was terminated with acid, and the absorbance value at 450 nm was measured on an ELISA reader.

[0078] Determination of the Specificity of Monoclonal Antibodies

[0079] The specificity of an antibody refers to its ability to recognize the corresponding antigen or a similar antigenic substance. The higher the specificity of the antibody, the stronger its recognition ability. Specificity is usually measured by the cross-reactivity rate, which can be determined by a competitive inhibition assay. Competitive inhibition curves are made with different concentrations of the antigen and the similar antigen, respectively, the binding rate of each is calculated, the concentration at IC50 of each is determined, and the cross-reactivity rate is calculated according to the following formula.

[0080] Cross-reactivity rate =

[0081] Calculated according to the above formula, the present invention tested the cross-reactivity rate of the antibody with other main components of cannabis: tetrahydrocannabinol (THC) < 1%, cannabinol (CBC) < 1%, cannabinol (CBN) < 1%, cannabigerol (CBG) < 1%. The antibody of the present invention has basically no cross-reactivity with tetrahydrocannabinol (THC), cannabinol (CBC), cannabinol (CBN), and cannabigerol (CBG), indicating that the antibody has good specificity.

[0082] 4. Preparation of Goat Anti-Mouse Secondary Antibody

[0083] Using sheep as the immunized animal, pathogen-free sheep are immunized with a mouse-derived antibody as the immunogen to obtain goat anti-mouse secondary antibody.

[0084] 5. Preparation of Gold-Labeled Antibody

[0085] (1) Preparation of Colloidal Gold

[0086] Dilute 1% chloroauric acid to 0.04% (mass fraction) with double-distilled deionized water, take 100 ml and place it in a conical flask, heat it to boiling with a thermostatic magnetic stirrer, add 10 ml of 1% trisodium citrate under continuous high temperature and continuous stirring, continue to stir and heat evenly until the solution turns bright red and then stop, cool to room temperature and restore the volume to the original volume with deionized water, and store at 4°C. The prepared colloidal gold is pure, transparent, without precipitation and floating matter.

[0087] (2) Preparation of Colloidal Gold-Labeled Cannabidiol Monoclonal Antibody

[0088] Take 5 ml of 0.04% colloidal gold, add 30 μl of 0.2 M K2CO3, stir for 5 min, add 25 μg of cannabidiol monoclonal antibody, stir for 5 min, then add 50 μl of 10% BSA to block and terminate the labeling; centrifuge at 10000 rpm for 7 min, discard the supernatant, resuspend the precipitate with colloidal gold reconstitution solution, and finally make up the volume to 0.5 ml with colloidal gold reconstitution solution (i.e., 1 / 10 of the volume of the colloidal gold solution).

[0089] Colloidal gold complex solution: 0.02 mol / L phosphate buffer containing 10% sucrose, 0.5% bovine serum albumin, and 0.1% Tween 20.

[0090] 6. Preparation of the conjugate release point

[0091] Dilute the colloidal gold-labeled cannabidiol monoclonal antibody with the colloidal gold complex solution to an appropriate multiple, and evenly spray it onto the conjugate pad at 1 μL / cm using a gold spraying instrument. Dry it at 37 °C for 12 h and store it in a dry environment (humidity < 20%) for later use.

[0092] 7. Preparation of the reaction membrane

[0093] Coat the reaction membrane with cannabidiol hapten-carrier protein conjugate and goat anti-mouse secondary antibody respectively: Adjust the cannabidiol hapten-carrier protein conjugate to 0.1 - 2 mg / mL with 0.02 M PBS at pH 7.4, and coat it on the NC membrane to form the test line, with a coating amount of 0.5 - 3 μL / cm; Adjust the goat anti-mouse secondary antibody to 0.02 - 1.0 mg / mL with 0.01 M PBS at pH 7.4, and coat it on the reaction membrane to form the quality control line, with a coating amount of 0.5 - 3 μL / cm. Place the coated reaction membrane in an oven at 37 °C for 1 - 2 h and store it in a dry environment for later use.

[0094] 8. Preparation of the sample absorption pad

[0095] Soak the sample absorption pad in a phosphate buffer containing 0.2% Tween 20, 0.5% PVP K30, 0.5% bovine serum albumin, pH 7.4, and 0.02 mol / L for 2 h, dry it in an oven at 37 °C for 24 h, and store it in a dry environment for later use.

[0096] 9. Assembly of the test strip

[0097] On the bottom plate, paste the sample absorption pad, conjugate release pad, reaction membrane, and absorbent pad in sequence. The sample absorption pad covers the conjugate release pad, and finally cut it into small strips 4 mm wide, put in a desiccant, seal it in an aluminum foil bag, and it can be stored for 12 months at 4 - 30 °C.

[0098] Example 2 Detection of the cannabidiol content in industrial hemp

[0099] 1. Sample pretreatment

[0100] 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 complex solution, mix well, and take 100 μL for analysis.

[0101] 2. Test strip detection

[0102] Use a pipette or a small dropper to take 100 μL of the diluted sample solution (4 - 5 drops with the dropper) and vertically drop it into the sample addition hole; start timing when the liquid starts to flow, and perform chromatography for 10 minutes, then interpret the result.

[0103] 3. Analysis of Test Results

[0104] Negative: If the color development of the test line (T) is darker than or the same as that of the control line (C), it indicates that the concentration of cannabidiol in the sample is lower than the detection limit, and it is judged as negative.

[0105] Positive: If the color development of the test line (T) is lighter than that of the control line (C) or the test line (T) does not show color, it indicates that the concentration of cannabidiol in the sample is equal to or higher than the detection limit, and it is judged as positive.

[0106] Invalid: When the control line (C) does not show a red band, the test strip is judged as invalid regardless of whether the test line (T) shows a red band or not.

Claims

1. A test strip for detecting cannabidiol, comprising a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate; Characterized in that: The conjugate release pad is sprayed with a colloidal gold-labeled cannabidiol-specific antibody, and the detection line and the quality control line are fixed on the reaction membrane. The detection line is coated with a cannabidiol hapten-carrier protein conjugate, and the quality control line is coated with goat anti-mouse secondary antibody; the cannabidiol-specific antibody is obtained by using a cannabidiol hapten-carrier protein conjugate as an immunogen. The cannabidiol hapten is prepared by the following method: Ethyl (2E,4E)-5-(3,5-dimethoxyphenyl)-2,4-pentadienoate is generated by the reaction of 3,5-dimethoxybenzaldehyde and triethyl-4-phosphide, and then reduced by PdC / H2, demethylated by HBr / HoAc and saponified to obtain 5-(3,5-dihydroxyphenyl)pentanoic acid, and finally reacted with (1S,4R)-1-methyl-4-(1-methylethenyl)-2-cyclohexen-1-ol; The specific reaction process is as follows: 。 2. The test strip for detecting cannabidiol according to claim 1, Characterized in that: The specific method and reaction process are as follows: 1) Take 0.87 g of 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 dropwise under an ice bath, stir and react for 30 min, and 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, and 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 and quench the reaction with 120 ml of water. Extract the aqueous phase with 120 ml of ethyl acetate 3 times, 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, that is, 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 with 150 ml of ethyl acetate 3 times, 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 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, add them to a 50 ml mixed solution of THF:DCM = 1:4, stir and mix evenly, and gradually 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 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, further purify it by preparative HPLC to obtain 220 mg of the CBD hapten.

3. The test strip according to claim 1, characterized in that: the sample absorption pad, the conjugate release pad, the reaction membrane, and the water absorption pad are sequentially pasted on the bottom plate.

4. The test strip according to claim 1 or 3, characterized in that: 1 / 3 - 1 / 2 of the conjugate release pad is covered under the sample absorption pad.

5. The test strip according to claim 1, characterized in that: the CBD hapten-carrier protein conjugate is obtained by conjugating the CBD hapten with a carrier protein, and the carrier protein is hemocyanin, bovine serum albumin, ovalbumin, human serum albumin or thyroprotein.

6. The test strip according to claim 1, characterized in that: the CBD-specific antibody is prepared using the CBD hapten-carrier protein conjugate as an immunogen.

7. The test strip according to claim 1, characterized in that: the goat anti-mouse secondary antibody is obtained by immunizing sheep with a murine antibody.

8. A method for preparing the test strip according to any one of claims 1-7, characterized in that: it includes the following steps: 1) Prepare a conjugate release pad coated with colloidal gold-labeled CBD-specific antibody; 2) Prepare a reaction membrane with a detection line coated with the CBD hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse secondary antibody; 3) Assemble the conjugate release pad and reaction membrane prepared in 1) and 2) with the sample absorption pad, the water absorption pad and the bottom plate into a test strip.

9. A method for detecting the CBD content in industrial hemp using the test strip according to any one of claims 1-7, characterized in that: the specific steps are as follows: 1) Pretreat the sample to be tested to obtain a sample solution to be tested; 2) Detect the sample solution to be tested with the test strip according to any one of claims 1-7; 3) Analyze the detection result.

Images