CN115327126A

CN115327126A - Time-resolved fluorescent microsphere immunochromatography test strip for detecting cannabidiol and preparation method and application thereof

Info

Publication number: CN115327126A
Application number: CN202210953074.1A
Authority: CN
Inventors: 吕小翠; 贾嘉; 袁利杰; 孟二娟; 赵琳; 夏静雪; 石小青; 陈丽丽; 崔浩哲; 纪元
Original assignee: Zhengzhou Zuoan Detection Technology Co ltd
Current assignee: Zhengzhou Zuoan Detection Technology Co ltd
Priority date: 2022-08-10
Filing date: 2022-08-10
Publication date: 2022-11-11

Abstract

A time-resolved fluorescent microsphere immunochromatographic test strip for detecting cannabidiol and a preparation method and application thereof are provided. The conjugate release pad is sprayed with a cannabidiol monoclonal antibody marked by a time-resolved fluorescent microsphere, a detection line and a 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 a goat anti-mouse secondary antibody. The cannabidiol monoclonal antibody is obtained by taking a cannabidiol-hapten carrier protein conjugate as an immunogen, and the cannabidiol hapten retains the characteristic structure of cannabidiol to the maximum extent. According to the invention, the antibody is labeled by the time-resolved fluorescent microspheres, and the immunochromatography technology is adopted, so that the rapid immunoassay of cannabidiol in a sample is realized, the sensitivity is high, the quantification is accurate, the detection is rapid, the operation is convenient, and the rapid detection and the field detection of a large batch of samples can be realized.

Description

Time-resolved fluorescent microsphere immunochromatographic test strip for detecting cannabidiol and preparation method and application thereof

Technical Field

The invention belongs to the technical field of test strip preparation and application, and particularly relates to a time-resolved fluorescence microsphere immunochromatography test strip for detecting cannabidiol, and a preparation method and application thereof.

Background

Cannabidiol, CBD, is a phytocannabinoid extracted from cannabis sativa, lacks psychotropic activity, and has analgesic, anti-inflammatory, anti-tumor and chemopreventive activities. Cannabidiol has the functions of blocking the adverse effect of certain polyphenol on the nervous system of a human body, blocking breast cancer metastasis, treating epilepsy, resisting rheumatoid arthritis, resisting insomnia and other physiological activities, and has good effect on treating multiple sclerosis.

Industrial hemp is hemp containing Tetrahydrocannabinol (THC) less than 0.3%, and is an annual herb plant of Cannabis (Cannabis) of Cannabaceae. The Cannabidiol (CBD) extracted from the flowers and leaves of the industrial cannabis sativa has wide market prospect, and the determination of the content of the cannabidiol in the industrial cannabis sativa is beneficial to monitoring the content of the CBD in the planting process of the industrial cannabis sativa and optimizing the variety of the industrial cannabis sativa.

At present, the conventional cannabidiol detection method mainly adopts an instrument method, such as high performance liquid chromatography and liquid chromatography tandem mass spectrometry, and the analysis method not only needs expensive instruments and professional technicians, but also has complex sample pretreatment process, long period and high cost, and is difficult to meet the requirements of rapid detection of a large number of samples and field samples. The immunoassay method based on antigen-antibody specific recognition can qualitatively and quantitatively detect the content of cannabidiol in a sample.

The time-resolved fluorescence immunochromatography technology adopts polystyrene nano-microspheres to wrap rare earth fluorescent ions, and the fluorescence intensity of a single fluorescent ion is improved through a fluorescence pre-enhancement technology. Through optimization and improvement of the wrapping technology, after the fluorescent ions are wrapped, glucan modification is carried out on the surfaces of the microspheres, so that the stability of the microspheres and the anti-interference performance to a reversible environment are greatly improved. On the basis, the developed fluorescence quantitative immunochromatographic technique has 1-3 orders of magnitude higher sensitivity than that of colloidal gold, colored latex and common fluorescence immunochromatographic methods, and has better stability and precision and wider linear range. Therefore, the immunochromatographic assay quantitative test strip based on time-resolved fluorescence has the advantages of high sensitivity, high flux, simplicity and rapidness.

Disclosure of Invention

The invention aims to provide a time-resolved fluorescent microsphere immunochromatographic test strip for detecting cannabidiol and a preparation method thereof based on the prior art, and provides a rapid, simple and efficient qualitative detection method suitable for mass samples.

The test strip 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 cannabidiol monoclonal antibody marked by a time-resolved fluorescent microsphere, a detection line and a quality control line are fixed on the reaction membrane, the detection line is coated with a cannabidiol hapten-carrier protein conjugate, the quality control line is coated with a goat anti-mouse secondary antibody, the cannabidiol monoclonal antibody is obtained by taking the cannabidiol hapten and the carrier protein conjugate as an immunogen, and the cannabidiol hapten is prepared by the following method: reacting 3, 5-dimethoxybenzaldehyde with triethyl-4-phosphide to produce ethyl (2E, 4E) -5- (3, 5-dimethoxyphenyl) -2, 4-pentadienoate, then carrying out PdC/H2 reduction, HBr/HoAc demethylation and saponification to obtain 5- (3, 5-dihydroxyphenyl) pentanoic acid, and finally reacting with (1S, 4R) -1-methyl-4- (1-methylvinyl) -2-cyclohexene-1-ol to obtain the cannabidiol hapten with the following structural formula:

the specific reaction process is shown in FIG. 4.

The carrier protein is hemocyanin, bovine serum albumin, ovalbumin, human serum albumin or thyroid protein.

The goat anti-mouse antibody is obtained by immunizing a goat with a murine antibody.

The sample absorption pad, the conjugate release pad, the reaction membrane and the water absorption pad are sequentially adhered to the base plate, and 1/3-1/2 of the conjugate release pad is covered under the sample absorption pad.

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

The time-resolved fluorescent microsphere is a fluorescent microsphere taking rare earth ions with the diameter of 100 nm-500 nm as an embedding material, the surface of the fluorescent microsphere is modified with functional groups, and the fluorescent microsphere is used for covalent coupling of protein, antibody and nucleic acid.

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

The method for preparing the test strip for detecting cannabidiol comprises the following steps:

1) Preparing a cannabidiol monoclonal antibody release pad marked by a spray-coating time-resolved fluorescent microsphere;

2) Preparing a reaction film with a detection line coated with a cannabidiol hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse secondary antibody.

3) And (3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a bottom plate to form the test strip.

Specifically, the steps include:

1) Hapten preparation: reacting 3, 5-dimethoxybenzaldehyde with triethyl-4-phosphide to produce ethyl (2E, 4E) -5- (3, 5-dimethoxyphenyl) -2, 4-pentadienoate, subsequently reducing by PdC/H2, HBr/HoAc demethylation and saponification to produce 5- (3, 5-dihydroxyphenyl) pentanoic acid, and finally reacting with (1S, 4R) -1-methyl-4- (1-methylvinyl) -2-cyclohexen-1-ol to produce cannabidiol hapten;

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

3) Immunizing a mouse by using a cannabidiol hapten-carrier protein conjugate, and fusing and screening splenocytes of the mouse and SP2/0 myeloma cells to obtain a cannabidiol hybridoma cell strain;

4) Extracting mouse IgG to immunize a healthy goat to obtain a goat anti-mouse secondary antibody;

5) Covalently coupling the cannabidiol monoclonal antibody prepared in the step 3) with a time-resolved fluorescent microsphere;

6) Spraying the cannabidiol monoclonal antibody marked by the time-resolved fluorescent microspheres on a conjugate release pad, drying at 37 ℃ for 12 hours, taking out, and storing in a dry environment for later use;

8) Coating the cannabidiol hapten-carrier protein conjugate on a reaction film to form a detection line, and coating a goat anti-mouse secondary antibody on the reaction film to form a quality control line;

9) Soaking the sample absorption pad in 0.2% Tween20, 0.5% PVP K30, 0.5% bovine serum albumin, pH7.4, 0.02mol/L phosphate buffer solution for 2h, oven drying at 37 deg.C for 24h, and storing in dry environment;

10 A sample absorption pad, a conjugate release pad, a reaction film and a water absorption pad are sequentially adhered on a bottom plate, the sample absorption pad covers the conjugate release pad, and finally the sample absorption pad is cut into small strips with the width of 4mm, a drying agent is put into the small strips, the small strips are placed in an aluminum foil bag for sealing, and the small strips can be stored for 12 months at the temperature of 4 to 30 ℃.

The invention also aims to provide a method for detecting cannabidiol in industrial cannabis sativa by using the test strip, which comprises the following steps:

(1) Pretreating a sample to be detected to obtain a sample solution to be detected;

(2) Detecting with a test strip;

(3) And inserting the detection card into an immunofluorescence analyzer, and calculating the concentration of cannabidiol in the sample by the analyzer through a built-in standard curve to analyze the detection result.

The cannabidiol rapid detection test strip adopts a highly specific antibody antigen reaction and immunochromatography analysis technology, the cannabidiol antibody of the time-resolved fluorescent microsphere is fixed on the conjugate release pad, and the cannabidiol in the sample is combined with the cannabidiol monoclonal antibody marked by the time-resolved fluorescent microsphere on the conjugate release pad in the flowing process to form the cannabidiol-antibody-time-resolved fluorescent microsphere marker. The cannabidiol in the sample competes with the cannabidiol hapten-carrier protein conjugate on the reaction membrane detection line to be combined with the cannabidiol monoclonal antibody marked by the time-resolved fluorescent microspheres, and the concentration of the cannabidiol is determined according to the ratio of the fluorescence on the detection line and the quality control line.

Compared with the prior art, the invention has the following advantages:

(1) The method can accurately quantify the content of the cannabidiol in the sample to be detected.

(2) The invention adopts the reaction systems of the independent quality control line and the detection line without mutual interference and influence, and adopts the T/C value mode for calibration, thereby ensuring the accuracy of the test result.

(3) The invention adopts the time-resolved fluorescent microspheres, and the Stokes displacement is large (more than 150 nm) and the fluorescence lifetime is 5 to 6 orders of magnitude higher than that of a background substance, so that the interference of various non-specific fluorescence can be effectively eliminated, and the detection sensitivity is improved.

(4) Fills the blank that no time-resolved fluorescent microsphere test strip for detecting cannabidiol exists at present.

(5) The cannabidiol hapten adopted by the invention not only furthest reserves the characteristic structure of cannabidiol, but also is coupled with carrier protein to form a connecting arm with a proper structure, so that the characteristic structure of cannabidiol is fully exposed to an organism as much as possible, the immune effect is enhanced, the specificity of an antibody is improved, and the cross reaction with other cannabidiol analogues is reduced;

(6) The carboxyl is introduced into the original structure of the cannabidiol hapten prepared by the method, so that the preparation of the cannabidiol artificial antigen is facilitated; the cannabidiol artificial antigen provided by the invention has strong immunogenicity, and is beneficial to stimulating an organism to complete immune response, so that a high-quality monoclonal antibody is obtained, and a core reagent is provided for establishment of a cannabidiol immunoassay method.

(7) The hapten and the cannabidiol to be detected have high overlapping degree of the framework structures, and the immunogenicity of the cannabidiol artificial antigen is effectively improved. The hapten and the carrier protein are coupled to form a connecting arm with a proper structure, so that the steric hindrance is reduced, the characteristic structure of the cannabidiol is fully exposed to the body as far as possible, the immune effect is enhanced, and the affinity of the antibody is further improved.

(8) The test strip provided by the invention has the advantages of high sensitivity, strong specificity, low cost, simplicity in operation, short detection time, no limitation of detection equipment, simplicity in storage and long quality guarantee period.

Drawings

FIG. 1: the test value of the test strip of the invention is compared with the test result of the chromatograph-a linear regression graph,

FIG. 2: the structure schematic diagram of the test strip for detecting cannabidiol of the invention,

in the figure: 1. A sample absorbing pad; 2. a conjugate release pad; 3. a reaction film; 4. a water absorbent pad; 5. a base plate; 6. detecting lines; 7. and (4) a quality control line.

FIG. 3: a test strip sample detection result schematic diagram;

FIG. 4: synthetic scheme for cannabidiol haptens;

FIG. 5: cannabidiol hapten nuclear magnetic resonance hydrogen spectroscopy;

FIG. 6: cannabidiol hapten nuclear magnetic resonance carbon spectrum;

FIG. 7: cannabidiol complete antigen uv absorption spectroscopy.

Detailed Description

The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. In addition, various changes or modifications may be made by those skilled in the art within the scope defined by the appended claims, and these changes or modifications should also fall within the scope of the invention.

Example 1 preparation of test strip of cannabidiol

The preparation method of the test strip mainly comprises the following steps:

1) Preparing a cannabidiol monoclonal antibody release pad sprayed with a time-resolved fluorescent microsphere marker;

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

The following distribution is detailed:

1. preparation of cannabidiol haptens

0.87g (22 mmol) of sodium hydride was added to 20ml of dry anhydrous tetrahydrofuran and stirred to form a suspension, 50ml of anhydrous tetrahydrofuran containing 5g of triethyl-4-phosphide (20 mmol) was added dropwise in ice bath and stirred for reaction for 30min. Subsequently, 50ml of anhydrous tetrahydrofuran containing 2.77g of 3, 5-dimethoxybenzaldehyde was added dropwise, the ice bath was removed, and the reaction was stirred at room temperature for 1 hour and then heated to 60 ℃ for 1 hour. After completion of the reaction by TLC, the reaction mixture was cooled to room temperature and quenched with 120ml of water, the aqueous phase was extracted 3 times with 120ml of ethyl acetate, the organic phases were combined, the organic phase was back-extracted with saturated brine, dried over anhydrous sodium sulfate overnight and rotary evaporated to give 4.0g of intermediate 1.

3.6g of intermediate 1 and 10% Pd-C (0.74 g) were added to 75ml of methanol, and the reaction was stirred at room temperature with addition of hydrogen for 2 hours, pd-C was filtered, and the filtrate was concentrated to give 3.5g of a pale yellow oily liquid (intermediate 2).

50ml of 40% hydrobromic acid and 50ml of glacial acetic acid are added into the intermediate 2, and the mixture is refluxed for 4h at 60 ℃. After TLC determined the reaction was complete, the reaction mixture was cooled to room temperature and 150ml of ice water was added. The reaction mixture was extracted 3 times with 150ml of ethyl acetate, the organic phase was back-extracted with saturated brine, dried over anhydrous sodium sulfate overnight, and the organic phase was concentrated to give a crude product as a black oil. The crude product was purified by column chromatography (ethyl acetate/hexane 1.

1g of intermediate 3 (4.75 mmol), 0.18g of p-TsOH (0.95 mmol) were added to 50ml THF: DCM (1.

Process for preparing cannabidiol haptens ¹ H NMR， ¹³ C NMR is shown in FIGS. 5 to 6.

2. Preparation of antigens

Preparing an immunity source: the cannabidiol hapten is coupled with bovine serum albumin to obtain the immune antigen.

20mg of hapten, 23mg of EDC and 14mg of NHS are taken and added into 1ml of dioxane to be dissolved completely to obtain solution A, and the solution A is stirred and reacted for 4 hours at room temperature. 50mg of bovine serum albumin was dissolved in 5ml of 10mM PBS under stirring to obtain solution B. The solution A is added into the solution B dropwise, and the reaction is stirred at 4 ℃ overnight. And dialyzing the protein solution with 10mM PBS for 3 days after the reaction is finished, and changing the solution once every 6 hours to obtain the cannabidiol-bovine serum albumin conjugate, namely the immune antigen CBD-BSA.

Preparation of coating source: the cannabidiol hapten is coupled with ovalbumin to obtain the coating antigen.

10mg of hapten, 1ml of anhydrous DMF, 12mg of EDC and 7mg of NHS are taken and dissolved completely to obtain solution A, and the solution A is stirred and reacted for 4 hours at room temperature. 20mg of ovalbumin was dissolved in 5ml of 10mM PBS under stirring to obtain solution B. The solution A is added into the solution B dropwise, and the reaction is stirred at 4 ℃ overnight. And dialyzing the protein solution with 10mM PBS for 3 days after the reaction is finished, and changing the solution once every 6h to obtain the cannabidiol-ovalbumin conjugate, namely the envelope antigen CBD-OVA.

Identification of cannabidiol complete antigen

The complete antigen adopts ultraviolet spectroscopy to identify the coupling result, and the coupling ratio is calculated by using the concentration of small molecules and protein in the conjugate. The maximum absorption peak of cannabidiol hapten-carrier protein is significantly changed compared with the maximum absorption peak of cannabidiol hapten and carrier protein, indicating that the preparation of cannabidiol-carrier protein is successful (see fig. 7). The hapten to BSA coupling ratio was calculated to be 25:1, coupling ratio to OVA of 16:1.

3. preparation of cannabidiol monoclonal antibody

Animal immunization: balb/c female mice of 6-8 weeks old are selected, the immune antigen and Freund's adjuvant are mixed and emulsified, and the mice generate antiserum according to the immune dose of 100 ug/mouse.

Cell fusion and subcloning: taking splenocytes of Balb/c mice which are successfully immunized, adjusting the ratio of myeloma cells SP2/0 to splenocytes of the immunized mice to be 1 to 5 to 1, and fusing the cells, measuring cell culture supernatants by a competitive ELISA method, and screening out appropriate positive holes. Cloning the positive hole by using a limiting dilution method until obtaining the hybridoma cell strain which stably secretes the monoclonal antibody.

Preparing and purifying monoclonal antibodies: selecting 10 to 11-week-old Balb/c female mice, injecting an adjuvant into the abdominal cavity of the Balb/c mice, treating the abdominal cavity for about 7 to 15 days by 0.3 to 0.5ml of each mouse, carrying out expanded culture on the screened hybridoma cell strain, and then inoculating the hybridoma cell strain into the abdominal cavity of the Balb/c mice. Mouse ascites after mouse death and extraction is centrifuged at 3000rpm for 10min. The ascites is first purified by ammonium sulfate precipitation and then purified by a Protein G column to obtain the final monoclonal antibody.

Potency assay for monoclonal antibodies

The titer of the antibody measured by the Elisa method is 1:600000.

in competition Elisa, cannabidiol-OVA coats an ELISA plate, cannabidiol standard substance and cannabidiol monoclonal antibody are added, incubation is carried out for 2h at 37 ℃, washing is carried out, goat anti-mouse secondary antibody marked by HRP is added, TMB color developing solution is added after washing, reaction is terminated by acid, and the absorbance value at 450nm is measured on an ELISA reader.

Monoclonal antibody specificity assay

The specificity of an antibody refers to the ability to recognize a corresponding antigen or a substance similar to an antigen. The antibody has high specificity and strong recognition capability. Specificity is measured in terms of cross-reactivity. The rate of cross-reactivity can be measured using a competitive inhibition assay. The competitive inhibition curves are respectively made by antigens with different concentrations and approximate antigens, the respective binding rates are calculated, the concentration of each at the IC50 is calculated, and the cross reaction rate is calculated according to the following formula.

Rate of cross reaction =

The present invention tests the cross-reactivity of the antibodies to other major components of cannabis, calculated according to the formula: the Tetrahydrocannabinol (THC) is less than 1 percent, the cannabichromene (CBC) is less than 1 percent, the Cannabinol (CBN) is less than 1 percent, the Cannabigerol (CBG) is less than 1 percent, and the antibody of the invention basically has no cross reaction on the Tetrahydrocannabinol (THC), the cannabichromene (CBC), the Cannabinol (CBN) and the Cannabigerol (CBG), thereby indicating that the antibody has better specificity.

4. Preparation of goat anti-mouse secondary antibody

The goat anti-mouse secondary antibody is obtained by taking a goat as an immune animal and taking a mouse antibody as an immunogen to immunize a goat without a pathogen.

5. Preparation of fluorescent microsphere markers

1mg of fluorescent microspheres was centrifuged at 15000 rpm for 10min, the pellet was collected and resuspended in 1mL of coupling buffer (50 mM boric acid pH 8.5). EDC and NHS were then added at a molar ratio of 1. Adding coupling buffer solution (50 mM boric acid pH8.5) for resuspension, adding 40-150ug 8 cannabidiol monoclonal antibody into the solution, mixing uniformly, stirring at room temperature for reaction for 2-4 h, centrifuging at 10000 rpm for 10min to remove supernatant, adding 1mL of sealing buffer solution, mixing uniformly, reacting at room temperature for 1-2h, washing with sealing buffer solution for 3 times, resuspending and precipitating with 0.02M PBS (containing 0.1-1% BSA and 0.1-5% trehalose) with pH7.4 to obtain the prepared cannabidiol monoclonal antibody marked by the time-resolved fluorescence microspheres, and placing at 4 ℃ for later use.

6. Preparation of time-resolved fluorescent microsphere conjugate release pad:

the conjugate release pad was soaked with 0.01-0.05M Tris-HCl buffer (containing 0.1-5% trehalose and 0.01-1% Tween-20) containing 0.1-0.5% bovine serum albumin (mass fraction), pH7.4 for 2h, and dried at 37 ℃ for 2h for use. Uniformly spraying the cannabidiol monoclonal antibody marked by the time-resolved fluorescent microspheres onto a conjugate release pad by using a film spraying instrument, spraying 1-9uL of the cannabidiol antibody marked by the fluorescent microspheres onto each 1 cm of the conjugate release pad, drying at 37 ℃ for 1-2h, and placing in a drying environment for later use.

7. Preparation of the reaction film

Coating the cannabidiol hapten-carrier protein conjugate and a goat anti-mouse secondary antibody on reaction membranes respectively: adjusting the cannabidiol hapten-carrier protein conjugate to 0.1-2mg/mL by using PBS (0.02M) with pH7.4, coating on an NC (NC) membrane to form a detection line, wherein the coating amount is 0.5-3uL/cm; adjusting the goat anti-mouse secondary antibody to 0.02-1.0mg/mL by using PBS (0.01M) with pH7.4, coating the secondary antibody on the reaction membrane to form a quality control line, wherein the coating amount is 0.5-3uL/cm. And (3) drying the coated reaction membrane at 37 ℃ for 1-2h, and placing the reaction membrane in a drying environment for later use.

8. Preparation of sample absorbent pad

Soaking the sample absorption pad in 0.2% Tween20, 0.5% PVP K30, 0.5% bovine serum albumin, pH7.4, 0.02mol/L phosphate buffer solution for 2h, oven drying at 37 deg.C for 24h, and storing in dry environment for use.

9. Assembly of test strips

And sequentially sticking a sample absorption pad, a conjugate release pad, a reaction film and a water absorption pad on the bottom plate, covering the conjugate release pad by the sample absorption pad, finally cutting into small strips with the width of 4mm, putting a drying agent into the small strips, sealing the small strips in an aluminum foil bag, and storing the small strips for 12 months at the temperature of 4 to 30 ℃.

Example 2 detection of cannabidiol content in Industrial hemp

1. Establishment of cannabidiol standard curve

Preparing cannabidiol standard substance with the sample diluent to the final concentration of 0, 0.05ug/mL, 0.10ug/mL, 0.25ug/mL, 0.50ug/mL, 1.00ug/mL, 2.5ug/mL, 5ug/mL, 10ug/mL, 20ug/mL, taking the test paper strip for detection, and repeating the determination for five times for each sample. The results of five replicates of the assay were averaged and then calibrated on an immunofluorescence analyzer.

2. Detection of cannabidiol content in industrial hemp sample

Pretreatment: weighing 10mg of industrial hemp sample into a 5mL grinding tube, adding 1mL of methanol, grinding for four minutes, standing, taking 50uL of supernatant, adding the supernatant into 950uL of complex solution, fully and uniformly mixing, and taking 100uL for analysis.

Taking 100uL (4-5 drops of a dropper) of the diluted sample liquid by using a pipette or a small dropper, and vertically dropping the diluted sample liquid into the sample adding hole; timing when the liquid flows, carrying out chromatography for 10min, reading the fluorescence intensity of the detection line and the quality control line by adopting an immunochromatography analyzer, giving a T/C value, and calculating the concentration of the cannabidiol in the sample by the analyzer through a built-in standard curve, and giving a detection result.

The test data of the test strip of the present invention is compared with the test result of the chromatograph, and the figure 1 is shown.

The chromatographic results and the test paper quantitative results of the industrial hemp samples are as follows:

Claims

1. a time-resolved fluorescent microsphere immunochromatographic test strip for detecting cannabidiol comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate; the method is characterized in that: the conjugate release pad is sprayed with a cannabidiol monoclonal antibody marked by a time-resolved fluorescent microsphere, a detection line and a quality control line are fixed on the reaction membrane, the detection line is coated with a cannabidiol hapten-carrier protein conjugate, the quality control line is coated with a goat anti-mouse secondary antibody, the cannabidiol monoclonal antibody is obtained by taking the cannabidiol hapten and the carrier protein conjugate as an immunogen, and the cannabidiol hapten is prepared by the following method: from 3, 5-dimethoxybenzaldehyde reacted with triethyl-4-phosphide to give ethyl (2E, 4E) -5- (3, 5-dimethoxyphenyl) -2, 4-pentadienoate, followed by PdC/H2 reduction, HBr/HoAc demethylation and saponification to give 5- (3, 5-dihydroxyphenyl) pentanoic acid, and finally reacted with (1S, 4R) -1-methyl-4- (1-methylvinyl) -2-cyclohexen-1-ol to give the cannabidiol hapten of the formula:

。

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

3. The immunochromatographic test strip according to claim 1, characterized in that: the time-resolved fluorescent microsphere is a fluorescent microsphere with rare earth ions with the diameter of 100-500 nm as an embedding material, the surface of the fluorescent microsphere is modified with functional groups, and the fluorescent microsphere is used for covalent coupling of protein, antibody and nucleic acid.

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

5. The immunochromatographic test strip according to claim 1, characterized in that: the cannabidiol hapten-carrier protein conjugate is obtained by coupling cannabidiol hapten and carrier protein, wherein the carrier protein is hemocyanin, bovine serum albumin, ovalbumin, human serum albumin or thyroid protein.

6. The immunochromatographic test strip according to claim 1, characterized in that: the conjugate release pad may be a glass fiber or a polyester fiber.

7. The immunochromatographic test strip according to claim 1, characterized in that: the specific reaction process for preparing cannabidiol hapten is as follows:

。

8. a method for preparing the immunochromatographic test strip of any one of claims 1 to 7, which is characterized in that: the method mainly comprises the following steps:

2) Preparing a reaction membrane with a detection line coated with a cannabidiol hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse secondary antibody;

9. A method for detecting the content of cannabidiol in industrial cannabis sativa by using the immunochromatographic test strip of any one of claims 1 to 7, which is characterized in that: the method mainly comprises the following steps:

pretreating a sample to be detected to obtain a sample solution to be detected;

detecting a sample solution to be tested with the test strip of any one of claims 1 to 7;

and inserting the detection card into an immunofluorescence analyzer, and calculating the concentration of cannabidiol in the sample by the analyzer through a built-in standard curve, and analyzing the detection result.