A time-resolved fluorescent microsphere immunochromatographic test strip for detecting cannabidiol and its preparation method and application

Abstract

A time-resolved fluorescence microsphere immunochromatographic 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 monoclonal antibody against cannabidiol labeled with time-resolved fluorescence microspheres. 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 monoclonal antibody against cannabidiol is obtained by using the cannabidiol-hapten carrier protein conjugate as an immunogen. The cannabidiol hapten retains the characteristic structure of cannabidiol to the greatest extent. The present invention uses time-resolved fluorescence microspheres to label antibodies and adopts immunochromatographic technology to realize rapid immunoassay of cannabidiol in samples, with high sensitivity, accurate quantification, rapid detection and convenient operation, and can realize rapid detection and on-site detection of a large number of samples.

Description

Technical Field

[0001] The present invention belongs to the technical field of test strip preparation and application, and specifically relates to a time-resolved fluorescence microsphere immunochromatographic test strip for detecting cannabidiol, a preparation method thereof, and an application thereof. 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 activity functions such as blocking breast cancer metastasis, treating epilepsy, anti-rheumatoid arthritis, anti-insomnia, etc., and also 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 CBD content in industrial hemp helps to monitor the CBD content during the cultivation process of industrial hemp and optimize 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. Immunological analysis methods based on the specific recognition of antigen and antibody can qualitatively and quantitatively detect the cannabidiol content in samples.

[0005] Time-resolved fluorescence immunochromatography technology uses polystyrene nanospheres to encapsulate rare earth fluorescent ions, and through fluorescence pre-enhancement technology, the fluorescence intensity of a single fluorescent ion is enhanced. Through the optimization and improvement of the encapsulation technology, after encapsulating the fluorescent ions, dextran modification is carried out on the surface of the microspheres, greatly improving the stability of the microspheres and the anti-interference ability against reversible environments. On this basis, the developed fluorescence quantitative immunochromatography technology has a sensitivity 1-3 orders of magnitude higher than that of colloidal gold, colored latex and ordinary fluorescence immunochromatography methods, better stability and precision, and a wider linear range. Therefore, the quantitative test strip based on time-resolved fluorescence immunochromatography technology has the advantages of high sensitivity, high throughput, simplicity and rapidity. Summary of the Invention

[0006] The purpose of the present invention is precisely based on the above-mentioned existing technical situation to provide a time-resolved fluorescence microsphere immunochromatographic test strip for detecting cannabidiol, a preparation method thereof, and to provide a rapid, simple and efficient qualitative detection method suitable for a large number of samples.

[0007] The test strip of the present invention comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate; wherein: the conjugate release pad is sprayed with a monoclonal antibody of cannabidiol labeled with time-resolved fluorescent microspheres, the reaction membrane is fixed with a test line and a quality control line, the test line is coated with a conjugate of cannabidiol hapten and carrier protein, the quality control line is coated with goat anti-mouse secondary antibody, the monoclonal antibody of cannabidiol is obtained by using a conjugate of cannabidiol hapten and carrier protein as an immunogen, and the cannabidiol hapten is prepared by the following method: ethyl (2E,4E)-5-(3,5-dimethoxyphenyl)-2,4-pentadienoate is generated by reacting 3,5-dimethoxybenzaldehyde with triethyl-4-phosphide, and then is reduced by PdC / H2, demethylated by HBr / HoAc and saponified to obtain 5-(3,5-dihydroxyphenyl) pentanoic acid, and finally reacts with (1S,4R)-1-methyl-4-(1-methylethenyl)-2-cyclohexen-1-ol to obtain, and the structural formula of the cannabidiol hapten is as follows:

[0008]

[0009] For the specific reaction process, see Figure 4 。

[0010] The carrier protein is hemocyanin, bovine serum albumin, ovalbumin, human serum albumin or thyroprotein.

[0011] The goat anti-mouse antibody is obtained by immunizing sheep with a murine 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 a cellulose acetate membrane.

[0014] The time-resolved fluorescent microspheres are fluorescent microspheres with rare earth ions with a diameter of 100 nm - 500 nm as the embedding material, and their surfaces are modified with functional groups for covalent coupling of proteins, antibodies and nucleic acids.

[0015] The excitation wavelength of the time-resolved fluorescent microspheres is 300 - 400 nm, and the emission wavelength is 500 - 700 nm.

[0016] A method for preparing the above test strip for detecting cannabidiol of the present invention comprises the following steps:

[0017] 1) Preparation of a release pad for cannabidiol monoclonal antibody labeled with time-resolved fluorescence microspheres;

[0018] 2) Preparation of 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.

[0019] 3) Assembling 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.

[0020] Specifically, the steps include:

[0021] 1) Hapten preparation: 3,5-dimethoxybenzaldehyde reacts with triethyl-4-phosphide to form 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. Finally, it reacts with (1S,4R)-1-methyl-4-(1-methylethenyl)-2-cyclohexen-1-ol to obtain the cannabidiol hapten;

[0022] 2) Coupling the cannabidiol hapten with a carrier protein to obtain a cannabidiol hapten-carrier protein conjugate;

[0023] 3) Immunizing mice with the cannabidiol hapten-carrier protein conjugate, and fusing and screening mouse spleen cells and SP2 / 0 myeloma cells to obtain a cannabidiol hybridoma cell line;

[0024] 4) Extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse secondary antibody;

[0025] 5) Covalently coupling the cannabidiol monoclonal antibody prepared in step 3) with time-resolved fluorescence microspheres;

[0026] 6) Spraying the time-resolved fluorescence microsphere-labeled cannabidiol monoclonal antibody on the conjugate release pad, taking it out after drying at 37°C for 12 h, and storing it in a dry environment for later use;

[0027] 8) Coating the reaction membrane with the cannabidiol hapten-carrier protein conjugate to form a test line, and coating the reaction membrane with goat anti-mouse secondary antibody to form a quality control line;

[0028] 9) Soaking the sample absorption pad in a phosphate buffer solution containing 0.2% Tween 20, 0.5% PVP K30, 0.5% bovine serum albumin, with a pH of 7.4 and a concentration of 0.02 mol / L for 2 h, drying it at 37°C for 24 h, and storing it in a dry environment for later use;

[0029] 10) Paste the sample absorption pad, conjugate release pad, reaction membrane, and water absorption pad on the bottom plate in sequence. The sample absorption pad covers the conjugate release pad. Finally, cut it into small strips with a width of 4 mm, put in a desiccant, place it in an aluminum foil bag and seal it. It can be stored for 12 months under the condition of 4 - 30 °C.

[0030] Another object of the present invention is to provide a method for detecting cannabidiol in industrial hemp by using the above test strip, which includes the steps:

[0031] (1) Pretreat the sample to be tested to obtain a sample solution to be tested;

[0032] (2) Detect with the test strip;

[0033] (3) Insert the test card into the immunofluorescence analyzer, and the analyzer can calculate the concentration of cannabidiol in the sample through the built-in standard curve and analyze the test result.

[0034] The rapid detection test strip for cannabidiol of the present invention adopts a highly specific antibody-antigen reaction and immunochromatographic analysis technology. The time-resolved fluorescence microsphere cannabidiol antibody is fixed on the conjugate release pad. During the flow of the cannabidiol in the sample, it binds to the cannabidiol monoclonal antibody labeled with time-resolved fluorescence microspheres on the conjugate release pad, forming a cannabidiol-antibody-time-resolved fluorescence microsphere label. The cannabidiol in the sample competes with the cannabidiol hapten-carrier protein conjugate on the detection line of the reaction membrane for binding to the cannabidiol monoclonal antibody labeled with time-resolved fluorescence microspheres, and the concentration of cannabidiol is determined according to the fluorescence ratio of the detection line to the quality control line.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] (1) The present invention can accurately quantify the content of cannabidiol in the sample to be tested.

[0037] (2) The present invention adopts a reaction system with independent quality control line and detection line, which do not interfere with each other, and is calibrated by the T / C value method, ensuring the accuracy of the test result.

[0038] (3) The present invention adopts time-resolved fluorescence microspheres. Because of its large Stokes shift (>150 nm) and the fluorescence lifetime being 5 - 6 orders of magnitude higher than that of the background substance fluorescence lifetime, it can effectively eliminate the interference of various non-specific fluorescence and improve the detection sensitivity.

[0039] (4) It fills the blank that there is currently no time-resolved fluorescence microsphere test strip for detecting cannabidiol.

[0040] (5) The CBD hapten adopted in the present invention not only retains the characteristic structure of CBD to the greatest extent, but also has a linker arm with a suitable structure in the conjugation with the carrier protein, enabling 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.

[0041] (6) The carboxyl group is introduced into the original structure of the CBD hapten prepared in the present invention, 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 conducive to stimulating the body to complete the immune response, thereby obtaining high-quality monoclonal antibodies and providing core reagents for the establishment of the CBD immunoassay method.

[0042] (7) 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. There is a linker arm with a suitable structure in the conjugation of the hapten with the carrier protein, reducing the steric hindrance, enabling 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.

[0043] (8) 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 : Comparison between the test values of the test strip of the present invention and the test results of the chromatograph - linear regression graph,

[0045] Figure 2 : Schematic structural diagram of a test strip for detecting CBD of the present invention,

[0046] In the figure: 1. Sample absorption pad; 2. Conjugate release pad; 3. Reaction membrane; 4. Absorbent pad; 5. Bottom plate; 6. Detection line; 7. Control line.

[0047] Figure 3 : Schematic diagram of the test results of the test strip sample;

[0048] Figure 4 : Synthetic route diagram of the CBD hapten;

[0049] Figure 5 : 1H NMR spectrum of the CBD hapten;

[0050] Figure 6 : 13C NMR spectrum of the CBD hapten;

[0051] Figure 7 : UV absorption spectrum of the CBD complete antigen. DETAILED DESCRIPTION OF THE INVENTION

[0052] 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 changes or modifications to the present invention within the scope defined by the appended claims, and these changes or modifications should also fall within the protection scope of the invention.

[0053] Example 1 Preparation of Cannabidiol Test Strip

[0054] The preparation method of the test strip mainly includes the following steps:

[0055] 1) Prepare a release pad sprayed with a time-resolved fluorescence microsphere-labeled cannabidiol monoclonal antibody;

[0056] 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.

[0057] 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.

[0058] The following will be described in detail:

[0059] 1. Preparation of Cannabidiol Hapten

[0060] 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. Subsequently, 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.

[0061] Add 3.6 g of intermediate 1 and 10% Pd-C (0.74 g) to 75 ml of methanol, add (pass) hydrogen and stir at room temperature for 2 h, filter Pd-C, concentrate the filtrate, and obtain 3.5 g of a pale yellow oily liquid (intermediate 2).

[0062] 50 mL of 40% hydrobromic acid and 50 mL of glacial acetic acid were added to Intermediate 2, and the mixture was refluxed at 60 °C for 4 h. After the reaction was determined to be complete by TLC, the reaction mixture was cooled to room temperature and 150 mL of ice water was added. The reaction mixture was extracted three times with 150 mL of ethyl acetate, and the organic phase was back-extracted with saturated brine, dried over anhydrous sodium sulfate overnight, and the organic phase was concentrated to obtain a crude product as a black oil. The crude product was purified by column chromatography (ethyl acetate / hexane 1:2) to obtain 2.50 g of Intermediate 3.

[0063] 1 g of Intermediate 3 (4.75 mmol) and 0.18 g of p-TsOH (0.95 mmol) were added to a 50 mL mixture of THF:DCM (1:4), 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 NaHCO3 solution. The organic phase was separated, washed twice with saturated brine, dried and concentrated to obtain 2.3 g of a crude product as an oil. After simple purification by column chromatography, it was further purified by preparative HPLC to obtain 220 mg of the CBD hapten.

[0064] For the CBD hapten 1 1H NMR, 13 13C NMR is as Figure 5-6 shown.

[0065] 2. Preparation of Antigen

[0066] Preparation of immunogen: The CBD hapten was conjugated with bovine serum albumin to obtain the immunogen.

[0067] 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 in 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 was completed, the protein solution was dialyzed against 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.

[0068] Preparation of coating antigen: The CBD hapten was conjugated with ovalbumin to obtain the coating antigen.

[0069] 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 by stirring with 5 ml of 10 mM PBS 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 coated antigen CBD-OVA.

[0070] Identification of the complete antigen of cannabidiol

[0071] The coupling result of the complete antigen was identified by ultraviolet spectroscopy, and the coupling 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 the maximum absorption peaks of the cannabidiol hapten and the carrier protein, indicating that the preparation of the cannabidiol-carrier protein was successful (see Figure 7 ). After calculation, the coupling ratio of the hapten to BSA was 25:1, and the coupling ratio to OVA was 16:1.

[0072] 3. Preparation of monoclonal antibody against cannabidiol

[0073] Animal immunization: Select 6-8-week-old female Balb / c mice, mix the immunizing antigen with Freund's adjuvant and emulsify it, and use an immunizing dose of 100 μg / mouse to produce antiserum in the mice.

[0074] Cell fusion and subcloning: Take the spleen cells of the successfully immunized Balb / c mice, adjust the ratio of myeloma cells SP2 / 0 to 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. Clone the positive wells using the limited dilution method until a hybridoma cell line that stably secretes monoclonal antibody is obtained.

[0075] Preparation and purification of monoclonal antibody: Select 10 - 11-week-old female Balb / c mice, inject adjuvant into the peritoneal cavity of the Balb / c mice, 0.3 - 0.5 ml per mouse, and treat the peritoneal cavity for about 7 - 15 days. Expand the culture of the screened hybridoma cell line and then inoculate it into the peritoneal cavity of the Balb / c mice. Sacrifice the mice by cervical dislocation and extract the ascites of the mice, and centrifuge at 3000 rpm for 10 min. The ascites is first purified by ammonium sulfate precipitation method and then purified by Protein G column to obtain the final monoclonal antibody.

[0076] Determination of the titer of monoclonal antibody

[0077] The titer of the antibody determined by the Elisa method was 1:600000.

[0078] Competitive ELISA: The ELISA plate was coated with cannabidiol-OVA. After adding the cannabidiol standard and the cannabidiol monoclonal antibody and incubating at 37 °C for 2 h, it was washed. Then, the HRP-labeled goat anti-mouse secondary antibody was added. After washing again, the TMB chromogenic solution was added, and the reaction was terminated with acid. The absorbance value at 450 nm was measured on an ELISA reader.

[0079] Determination of Monoclonal Antibody Specificity

[0080] The specificity of an antibody refers to its ability to recognize the corresponding antigen or an antigenically similar substance. The higher the specificity of an 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 antigenically similar substance, 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.

[0081] Cross-reactivity rate =

[0082] Calculated according to the above formula, the cross-reactivity rate of the antibody of the present invention with other main components of cannabis was tested: tetrahydrocannabinol (THC) < 1%, cannabinol (CBC) < 1%, cannabinol (CBN) < 1%, cannabigerol (CBG) < 1%. The antibody of the present invention showed basically no cross-reactivity with tetrahydrocannabinol (THC), cannabinol (CBC), cannabinol (CBN), and cannabigerol (CBG), indicating good antibody specificity.

[0083] 4. Preparation of Goat Anti-Mouse Secondary Antibody

[0084] Using goats as the immunized animals, pathogen-free goats were immunized with a mouse-derived antibody as the immunogen to obtain the goat anti-mouse secondary antibody.

[0085] 5. Preparation of Fluorescent Microsphere Label

[0086] Take 1 mg of fluorescent microspheres and centrifuge them at 15,000 rpm for 10 min. Collect the precipitate and resuspend it in 1 mL of coupling buffer (50 mM boric acid, pH 8.5). Then add EDC and NHS at a molar ratio of 1:2 - 1:20 respectively. After vortexing, incubate at room temperature for 20 - 30 min, and then centrifuge at 15,000 rpm for 10 min to collect the precipitate. Add coupling buffer (50 mM boric acid, pH 8.5) to resuspend the microspheres. Add 40 - 150 μg of cannabidiol monoclonal antibody to this solution, mix well, and stir at room temperature for 2 - 4 h. Centrifuge at 10,000 rpm for 10 min to remove the supernatant. Add 1 mL of blocking buffer, mix well, and react at room temperature for 1 - 2 h. After centrifugally washing 3 times with blocking buffer, resuspend the precipitate in 0.02 M PBS (pH 7.4) containing 0.1 - 1% BSA and 0.1 - 5% trehalose to obtain the prepared time-resolved fluorescent microsphere-labeled cannabidiol monoclonal antibody, which is stored at 4°C for later use.

[0087] 6. Preparation of the conjugate release pad for time-resolved fluorescent microspheres:

[0088] Soak the conjugate release pad in a buffer containing 0.1 - 0.5% bovine serum albumin (mass fraction), 0.01 - 0.05 M Tris-HCl (pH 7.4) containing 0.1 - 5% trehalose and 0.01 - 1% Tween-20 for 2 h, and then dry it at 37°C for 2 h for later use. Use a film spraying instrument to evenly spray the time-resolved fluorescent microsphere-labeled cannabidiol monoclonal antibody onto the conjugate release pad. Spray 1 - 9 μL of the fluorescent microsphere-labeled cannabidiol antibody per 1 cm of the conjugate release pad, and dry it at 37°C for 1 - 2 h, then store it in a dry environment for later use.

[0089] 7. Preparation of the reaction membrane

[0090] Coat the cannabidiol hapten-carrier protein conjugate and goat anti-mouse secondary antibody on the reaction membrane respectively: Adjust the cannabidiol hapten-carrier protein conjugate to 0.1 - 2 mg / mL with 0.02 M PBS (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 (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 at 37°C and dry it for 1 - 2 h, then store it in a dry environment for later use.

[0091] 8. Preparation of the sample absorption pad

[0092] Soak the sample absorption pad in a phosphate buffer solution with 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.

[0093] 9. Assembly of the test strip

[0094] Paste the sample absorption pad, conjugate release pad, reaction membrane, and water absorption pad on the bottom 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.

[0095] Example 2 Detection of cannabidiol content in industrial hemp

[0096] 1. Establishment of the cannabidiol standard curve

[0097] Prepare cannabidiol standard products with the sample diluent to final concentrations of 0, 0.05 μg / mL, 0.10 μg / mL, 0.25 μg / mL, 0.50 μg / mL, 1.00 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL. Take the test strip for detection, and repeat the determination five times for each sample. After taking the average of the five repeated determination results, calibrate it on an immunofluorescence analyzer.

[0098] 2. Detection of cannabidiol content in industrial hemp samples

[0099] Pretreatment: 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 100 μL for analysis.

[0100] Use a pipette or a small dropper to vertically drop 100 μL of the diluted sample solution (4 - 5 drops of the dropper) into the sample addition hole; start timing when the liquid flows, chromatograph for 10 min, use an immunochromatographic analyzer to read the fluorescence intensities of the test line and the control line, and give the T / C value. The analyzer can calculate the cannabidiol concentration in the sample through the built-in standard curve and give the test result.

[0101] For the comparison of the test data of the test strip of the present invention with the test results of the chromatograph, see Figure 1 .

[0102] The chromatographic results and the quantitative results of the test strip for industrial hemp samples are as follows:

[0103]

Claims

1. A time-resolved fluorescence microsphere immunochromatographic test strip for detecting cannabidiol, the test strip comprises: 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 monoclonal antibody against cannabidiol labeled with time-resolved fluorescence microspheres, 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 monoclonal antibody against cannabidiol is obtained by using a cannabidiol hapten-carrier protein conjugate as an immunogen, and the cannabidiol hapten is prepared by the following method: (2E,4E)-5-(3,5-dimethoxyphenyl)-2,4-pentadienoic acid ethyl ester 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)valeric acid, and finally reacted with (1S,4R)-1-methyl-4-(1-methylethenyl)-2-cyclohexen-1-ol, and the structural formula of the cannabidiol hapten is as follows: The specific reaction process is as follows: 。 2. The immunochromatographic test strip 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 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, 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 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 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, 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 NaHCO3 solution, separate the organic phase, wash it twice with saturated brine, dry and concentrate to obtain 2.3 g of oily crude product. After simple purification by column chromatography and further purification by preparative HPLC, 220 mg of the cannabidiol hapten is obtained.

3. The immunochromatographic test strip according to claim 1, characterized in that: The reaction membrane can be a nitrocellulose membrane or a cellulose acetate membrane.

4. The immunochromatographic test strip according to claim 1, characterized in that: The time-resolved fluorescence microspheres are fluorescence microspheres with rare earth ions with a diameter of 100 nm - 500 nm as the embedding substance, and their surfaces are modified with functional groups for covalent coupling of proteins and nucleic acids.

5. The immunochromatographic test strip according to claim 1, characterized in that: The excitation wavelength of the time-resolved fluorescence microspheres is 300 - 400 nm, and the emission wavelength is 500 - 700 nm.

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

7. The immunochromatographic test strip according to claim 1, characterized in that: The conjugate release pad can be glass fiber or polyester fiber.

8. A method for the immunochromatographic test strip according to any one of claims 1 - 7, characterized in that: comprises the following steps: 1) Prepare a conjugate release pad sprayed with time-resolved fluorescence microsphere-labeled cannabidiol monoclonal antibody; 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; 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.

9. A method for detecting the content of cannabidiol in industrial hemp by the immunochromatographic test strip according to any one of claims 1 - 7, characterized in that: comprises the following steps: 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) Insert the test card into an immunofluorescence analyzer, and the analyzer can calculate the concentration of cannabidiol in the sample through a built-in standard curve and analyze the test results.

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