Preparation and Application of a Cannabinoid Monoclonal Antibody

Abstract

The present invention discloses the preparation and application of a cannabinoid monoclonal antibody, belonging to the field of biotechnology. The specific steps for the preparation of the cannabinoid monoclonal antibody include: (1) hapten preparation: reacting a cannabinoid molecule with methyl 4-chlorobenzoate to obtain the hapten; (2) complete antigen preparation: coupling the hapten with hemocyanin to obtain the complete antigen. The present invention prepares the complete antigens of tetrahydrocannabinol, cannabigerol or cannabidiol, further prepares the corresponding monoclonal antibodies, and then uses the prepared monoclonal antibodies to prepare a colloidal gold test strip. The prepared colloidal gold test strip has strong specificity and high sensitivity. The complete antigen is obtained by coupling a tetrahydrocannabinol, cannabigerol or cannabidiol molecule with hemocyanin.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the preparation and application of a cannabinoid monoclonal antibody. Background Art

[0002] Cannabis is an ancient cultivated plant with medicinal development value. Cannabinoids are a general term for phenolic derivatives extracted from cannabis plants, which mainly contain tetrahydrocannabinol (THC), cannabinol (CBG), cannabidiol (CBD), tetrahydrocannabinolic acid, etc.

[0003] CBD is a monomer extracted from cannabis and is the first cannabis-based drug approved by the FDA. CBD is a non-addictive component in cannabis and has pharmacological effects such as antispasmodic, anti-anxiety, anti-inflammatory, antioxidant, anti-rheumatic, and anti-tumor effects.

[0004] CBG is a non-psychoactive cannabinoid. Research shows that CBG is very active in protecting brain nerve cells. In 2015, researchers from the Department of Biochemistry and Molecular Biology at Complutense University of Madrid, Spain, found that CBG has neuroprotective properties. CBG can improve motor deficits and preserve neurons, increase the level of antioxidant defense, and has a positive impact on neurodegenerative diseases such as multiple sclerosis and Huntington's disease.

[0005] THC is the main psychoactive substance in cannabis and is listed in the first category of psychotropic substances control list. Since THC threatens human health. While CBG and CBD have beneficial pharmacological effects, the rapid detection of various components in cannabis plants is a key technology for distinguishing high-quality industrial cannabis from drug cannabis. The rapid detection of THC can meet the needs of drug detection.

[0006] Common preliminary screening methods for detecting whether CBD, CBG, or THC is contained in a test sample include radioimmunoassay, enzyme-linked immunosorbent assay, colloidal gold immunochromatography technique (GICA), etc. The confirmation methods mainly use physical and chemical detection methods such as high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC / MS). Both HPLC and GC / MS tests must be completed in a laboratory, require equipment and are operated by professional technicians, and are expensive. Radioimmunoassay has high sensitivity and specificity, but the instrument is expensive, so it cannot be popularized. Enzyme-linked immunosorbent assay retains the advantages of radioimmunoassay in terms of sensitivity and specificity, but the operation is complex, time-consuming, and requires professional technicians to operate. Colloidal gold immunochromatography is very suitable for on-site screening of a large number of samples due to its advantages such as simple and fast, clear results, no need for complex operation skills and special equipment, high sensitivity and specificity, and has become a new direction for the development of the clinical diagnosis field.

[0007] There is an urgent need in this field to develop a simple, rapid, accurate, reliable, sensitive and inexpensive detection method applicable to agricultural planting detection, industrial hemp product detection and cannabis drug detection. Chinese Patent CN101580544A discloses a colloidal gold-labeled monoclonal antibody immunoassay strip for cannabis and tetrahydrocannabinol, which has the advantages of being able to detect tetrahydrocannabinol quickly, simply and sensitively, but this patent does not further study and test other cannabinoid molecules. Summary of the Invention

[0008] The first object of the present invention is to provide a cannabinoid complete antigen and a preparation method thereof. To achieve the object of the invention, the following technical solutions are adopted:

[0009] A cannabinoid hapten represented by Formula I or Formula II or Formula III,

[0010]

[0011] A cannabinoid complete antigen represented by Formula IV or Formula V or Formula VI,

[0012] In each formula, KLH is hemocyanin.

[0013] A method for the above-mentioned cannabinoid complete antigen, characterized by comprising the following steps:

[0014] (1) Preparation of hapten: Reacting a cannabinoid molecule with methyl 4-chlorobenzoate to obtain the hapten;

[0015] (2) Preparation of complete antigen: Coupling the hapten with hemocyanin (KLH) to obtain the complete antigen.

[0016] The immunogenicity of the cannabinoid molecule is weak. After coupling the cannabinoid molecule with a carrier protein, an antigen with stronger immunogenicity can be formed; and because there are no active groups on the surfaces of THC, CBG or CBD molecules that can be directly coupled to the protein, derivatization treatment is required to prepare a hapten before coupling the carrier protein to introduce an active carboxyl group.

[0017] Preferably, the specific operation steps of step (1) for preparing the hapten include:

[0018] Dissolving the cannabinoid molecule and a catalyst in DMF and stirring, then adding methyl 4-chlorobenzoate and heating and stirring for reaction; after the reaction, adding a LiOH solution for hydrolysis reaction; then extracting with ethyl acetate, collecting the organic phase, filtering and drying to obtain the hapten.

[0019] More preferably, the cannabinoid molecule is selected from tetrahydrocannabinol (THC), cannabigerol (CBG) or cannabidiol (CBD).

[0020] More preferably, the catalyst includes triethylamine, DMAP and NaOH.

[0021] Even more preferably, the weight ratio of triethylamine, DMAP and NaOH is: 5 - 10:6 - 8:1 - 2.

[0022] More preferably, the weight ratio of methyl 4 - chlorobenzoate to cannabinoid molecules is 2 - 3:4 - 8.

[0023] More preferably, the reaction temperature of methyl 4 - chlorobenzoate and cannabinoid molecules is 65 - 80 °C.

[0024] More preferably, the reaction time of methyl 4 - chlorobenzoate and cannabinoid molecules is 2 - 5 h.

[0025] More preferably, the concentration of LiOH is 20 - 30 wt%.

[0026] More preferably, the hydrolysis reaction time is 2 - 5 h.

[0027] More preferably, the hydrolysis reaction temperature is room temperature.

[0028] Even more preferably, the specific operation steps of the hapten in step (1) include:

[0029] By weight, add 4 - 8 parts of cannabinoid molecules, 5 - 10 parts of triethylamine, 6 - 8 parts of DMAP and 1 - 2 parts of NaOH to 15 - 20 parts of DMF and stir to dissolve. Then add 2 - 3 parts of methyl 4 - chlorobenzoate, heat to 65 - 80 °C and stir - react for 2 - 5 h; after the reaction, add 15 - 20 parts of water and extract with ethyl acetate; collect the organic phase, concentrate and dry, then add 15 - 20 parts of 20% - 30% LiOH solution, and carry out hydrolysis reaction at room temperature for 2 - 5 h; then extract with ethyl acetate, collect the organic phase and adjust the pH to 5 - 6, filter by suction and dry to obtain the hapten.

[0030] Preferably, the specific operation steps of preparing the complete antigen in step (2) include:

[0031] Dissolve hemocyanin in a buffer solution to obtain a hemocyanin solution; dissolve the hapten in DMF and add EDC and NHS; drop - wise add the hapten solution to the hemocyanin solution, and stir - react after dropping; after the reaction, dialyze to obtain the complete antigen.

[0032] More preferably, the buffer solution is CBS buffer solution, with pH 9.5 - 9.8 and concentration 0.05 - 0.1 M.

[0033] More preferably, the weight ratio of hemocyanin to the buffer solution is 2 - 8:15 - 30.

[0034] More preferably, the weight ratio of hemocyanin to hapten is 2-8:1-2.

[0035] More preferably, the weight ratio of hapten, DMF solution, EDC, and NHS is: 1-2:80-100:2-3:1-2.

[0036] More preferably, the reaction temperature is room temperature.

[0037] More preferably, the reaction time is 10-15h.

[0038] More preferably, the dialysis system is PBS buffer, pH 9-10, concentration 0.1-0.5M.

[0039] Even more preferably, the specific operation steps for preparing the complete antigen in step (2) include:

[0040] By weight, dissolve 2-8 parts of hemocyanin in 20-30 parts of CBS buffer (pH 9.5-9.8, concentration 0.05-0.1M); dissolve 1-2 parts of hapten in 80-100 parts of DMF, and add 2-3 parts of EDC and 1-2 parts of NHS; add the hapten solution dropwise to the hemocyanin solution, and stir and react at room temperature for 10-15h after dropping; after the reaction, dialyze with PBS buffer (pH 9-10, concentration 0.1-0.5M) to obtain the complete antigen.

[0041] The above method for preparing the complete antigen is only a preferred preparation method. In the preparation of the complete antigen of the present invention, any known prior art in the art can be used for conjugation.

[0042] Preferably, a method for preparing a cannabinoid monoclonal antibody further includes the following preparation steps:

[0043] Serum titer detection and screening; hybridoma cell preparation; hybridoma cell screening; in vivo induction in animals; antibody purification.

[0044] The present invention also discloses a cannabinoid monoclonal antibody prepared by the above preparation method.

[0045] The second object of the present invention is to provide a colloidal gold test strip that can accurately, quickly, and sensitively detect whether the sample contains THC, CBD, or CBG.

[0046] A colloidal gold test strip contains a cannabinoid monoclonal antibody.

[0047] Preferably, the cannabinoids include THC, CBD, or CBG. Most of the colloidal gold test strips in the prior art are used to detect THC. The colloidal gold test strip prepared by the present invention can simultaneously detect one or several of THC, CBD, or CBG, with a wider detection range, which is beneficial to improving the detection accuracy.

[0048] Preferably, the preparation method of the colloidal gold test strip comprises the following steps:

[0049] (1) Nanogold-labeled antibody: Mix the nanogold solution and the antibody and shake, add the blocking solution for blocking and then centrifuge, and resuspend to obtain the antibody suspension;

[0050] (2) Detection line preparation: Coat the antibody suspension at one end of the nitrocellulose membrane close to the sample pad to obtain the detection line;

[0051] (3) Quality control line preparation: Coat the goat anti-mouse antibody at one end of the nitrocellulose membrane close to the absorbent pad to obtain the quality control line;

[0052] (4) Test strip assembly: Stick the sample pad, the absorbent pad, the bottom plate, and assemble the nitrocellulose membrane to obtain the colloidal gold test strip.

[0053] More preferably, (4) the test strip assembly includes: Stick the sample pad and the absorbent pad at both ends of the bottom plate, stick the nitrocellulose membrane in the middle of the bottom plate and overlap with the sample pad and the absorbent pad, and after assembly, obtain the colloidal gold test strip.

[0054] More preferably, the antibody concentration in the antibody solution is 0.2 - 0.5 mg / mL.

[0055] More preferably, there are 1 - 3 detection lines, which are 1 or 3 of the THC detection line, the CBD detection line, or the CBG detection line. Therefore, the test strip of the present invention can detect whether the sample contains one or several of THC, CBD, or CBG alone or simultaneously. When detecting 2 or 3 kinds simultaneously, the detection accuracy can be increased.

[0056] More preferably, the steps of (1) nanogold-labeled antibody are as follows:

[0057] Mix the nanogold solution, the antibody, diglycine, and ethanolamine hydrochloride and shake, add the blocking solution for blocking and then centrifuge, and resuspend to obtain the antibody suspension.

[0058] The addition of diglycine and ethanolamine hydrochloride will not have a negative impact on the detection function of the test strip. On the contrary, it can also increase the stability of the colloidal gold and the antibody, enabling it to withstand higher temperatures. It is beneficial to avoid the test strip from failing due to high temperature.

[0059] Preferably, the weight ratio of diglycine to ethanolamine hydrochloride is 1 - 3:0.3 - 0.8.

[0060] The present invention also discloses the use of the above-mentioned complete antigen in the preparation of cannabinoid monoclonal antibodies.

[0061] The present invention also discloses the use of the above-mentioned diglycine and ethanolamine hydrochloride in the high-temperature resistance of the colloidal gold test strip.

[0062] The present invention also discloses the use of the above colloidal gold test strip in detecting whether a sample contains cannabinoid molecules.

[0063] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0064] By preparing the complete antigens of THC, CBD, and CBG, further preparing the monoclonal antibodies of THC, CBD, and CBG, and then using the prepared monoclonal antibodies to prepare a colloidal gold test strip, the obtained colloidal gold test strip has strong specificity and can accurately detect whether one or several of THC, CBD, or CBG are contained in the sample; it has high sensitivity, and the lowest detection concentration is at least 10 ng / mL; moreover, the colloidal gold test strip of the present invention can obtain the detection result within 90 s, and the detection speed is fast. When preparing the colloidal gold test strip of the present invention, glycylglycine and ethanolamine hydrochloride are also added to the antibody suspension prepared by labeling the antibody with nano-gold. When the nano-gold solution, glycylglycine, and ethanolamine hydrochloride are used together, it can increase the high-temperature resistance of the colloidal gold test strip and still be able to accurately detect after storing at 70 °C for 5 - 10 d. Description of the Drawings

[0065] Figure 1 It is the assembly sequence of the colloidal gold test strip. Detailed Embodiments

[0066] Here, the exemplary embodiments will be described in detail. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present disclosure. On the contrary, they are merely examples of methods consistent with some aspects of the present disclosure as detailed in the appended claims.

[0067] Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods or are carried out according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels.

[0068] BALB / c mice were purchased from MouseLab (Wuhan) Biotechnology Co., Ltd.; SP2 / 0 myeloma cells were purchased from Wuhan Punosai Life Technology Co., Ltd.

[0069] Example 1

[0070] Preparation of Complete Antigens

[0071] 1. Preparation of CBG Complete Antigen

[0072] (1) Preparation of CBG Hapten:

[0073] Dissolve 500 mg of CBG, 500 mg of triethylamine, 600 mg of DMAP, and 200 mg of NaOH in 2000 mg of DMF by stirring. Then add 300 mg of methyl 4-chlorobenzoate and heat to 75 °C, stirring and reacting for 3 h. After the reaction, add 1500 mg of water and extract with ethyl acetate. Collect the organic phase, concentrate and dry it, then add 2000 mg of 25% LiOH solution and carry out hydrolysis reaction at room temperature for 3.5 h. Then extract with ethyl acetate, collect the organic phase, adjust the pH to 5.5, filter by suction and dry to obtain the CBG hapten. The prepared hapten was characterized by 1H NMR, and the characterization results are as follows. It can be seen that the CBG hapten shown in Formula I was successfully prepared; 1 H NMR(400MHz,DMSO)δ:9.94(s,1H),8.19(d,2H),7.38(d,2H),6.70(s,1H),6.65(s,1H),5.82(d,1H),5.40(s,1H),5.29(m,1H),3.25(d,2H),2.67(m,2H),2.06(m,2H),2.00(d,2H),1.85(s,6H),1.72(s,3H),1.62(m,2H),1.30 - 1.32(m,4H,CH2),0.95(m,3H).

[0074]

[0075] (2) Preparation of complete antigen:

[0076] Dissolve 5 mg of hemocyanin in 25 mg of CBS buffer (pH 9.6, concentration 0.1 M) to obtain a hemocyanin solution; dissolve 2 mg of CBG hapten in 100 mg of DMF, and add 3 mg of EDC and 2 mg of NHS to obtain a CBG hapten solution; add the CBG hapten solution dropwise to the hemocyanin solution, and stir and react at room temperature for 10 h after dropping. After the reaction, dialyze with PBS buffer (pH 9.6, concentration 0.1 M) to obtain the complete antigen CBG-KLH shown in Formula IV.

[0077]

[0078] 2. Preparation of CBD complete antigen

[0079] (1) Preparation of CBD hapten:

[0080] The preparation method is the same as that of the CBG hapten to obtain the CBD hapten shown in Formula II. The nuclear magnetic structure of the CBD hapten shown in Formula II was characterized by a nuclear magnetic resonance instrument, and the nuclear magnetic resonance identification result is: 11H NMR (400 MHz, DMSO) δ: 9.89 (s, 1H), 8.19 (d, 2H), 7.38 (d, 2H), 6.74 (s, 1H), 6.69 (s, 1H), 5.40 (s, 1H), 5.35 (m, 1H), 5.14 (m, 1H), 4.95 (m, 1H), 3.25 (d, 1H), 2.75 (d, 1H), 2.67 (m, 2H), 2.06 (m, 1H), 2.00 (m, 1H), 1.85 (s, 6H), 1.79 (m, 1H), 1.58 (m, 2H), 1.52 (m, 1H), 1.30 - 1.32 (m, 4H), 0.95 (m, 3H).

[0081]

[0082] (2) Preparation of complete antigen:

[0083] The preparation method is the same as that of the CBG complete antigen, and finally CBD-KLH shown in Formula V is obtained.

[0084]

[0085] 3. Preparation of THC complete antigen

[0086] (1) Preparation of THC hapten:

[0087] The preparation method is the same as that of the CBG hapten, and the THC hapten shown in Formula III is obtained. The nuclear magnetic resonance structure of the THC hapten shown in Formula III is characterized by a nuclear magnetic resonance instrument, and the nuclear magnetic resonance identification result is: 1 1H NMR (400 MHz, DMSO) δ: 9.86 (s, 1H), 8.23 (d, 2H), 7.44 (d, 2H), 6.81 (s, 2H), 5.42 (d, 1H), 3.28 (m, 1H), 2.66 (m, 2H), 2.50 (t, 1H), 1.93 - 1.99 (t, 2H), 1.85 (s, 3H), 1.79 (t, 1H), 1.61 (m, 2H), 1.52 (t, 1H), 1.47 (s, 6H), 1.28 - 1.30 (m, 4H), 0.95 (m, 3H).

[0088]

[0089] (2) Preparation of complete antigen:

[0090] The preparation method is the same as that of the CBG complete antigen, and finally THC-KLH shown in Formula VI is obtained.

[0091]

[0092] Example 2

[0093] Preparation of Monoclonal Antibody

[0094] 1. Preparation of CBG Monoclonal Antibody

[0095] (1) Animal Immunization

[0096] Eight BALB / c mice numbered 1 - 8 were taken and immunized by mixing CBG - KLH with Freund's adjuvant. The weight ratio of CBG - KLH to Freund's adjuvant was 1:1. Immunization was carried out at a subcutaneous injection dose of 50 μg / mouse, and booster immunization was performed once every 3 weeks with the dose halved during booster immunization. A total of 3 immunizations were carried out. Blood was collected from the mouse tails on the 7th day after each immunization to measure the serum titer of the mice.

[0097] (2) Detection and Screening of Mouse Serum Titer

[0098] Coating the Plate: Dilute the antigen D - BSA to a concentration of 1 μg / mL with PBS coating solution (pH 7.4), mix well and add 100 μL to each well of a 96 - well plate, and incubate overnight in a 4°C refrigerator;

[0099] Blocking: After coating, discard the coating solution, wash the plate 3 times, add 200 μL of blocking solution to each well, and incubate in a 37°C incubator for 1 h; Take out the 96 - well plate, discard the internal solution, and wash the plate once;

[0100] Primary Antibody: Dilute the antiserum at different multiples starting from 1:500, add 100 μL to each well; The control is 0.01 M PBS; Incubate in a 37°C incubator for 1 h;

[0101] Secondary Antibody: Take out the 96 - well plate, discard the internal solution, wash the plate 3 times, and add 100 μL of goat anti - mouse - HRP diluted 1:20000 to each well; Incubate in a 37°C incubator for 1 h;

[0102] Color Development: Take out the enzyme - labeled plate, discard the internal solution, wash the plate 4 times, add 100 μL of TMB color - developing solution to each well first, and incubate in a 37°C incubator for 15 min;

[0103] Termination: Add 100 μL of 1 M HCL solution to each well to terminate the reaction; Immediately read the absorbance at 450 nm on an enzyme - labeled immunosorbent assay reader. The dilution corresponding to the well with an OD value greater than 2.1 times the OD value of the set negative control is defined as the titer of the sample;

[0104] The detection results are shown in Table 1;

[0105] Table 1 Detection of CBG Monoclonal Antibody Titer 7 Days after the Third Immunization

[0106]

[0107] According to Table 1, the serum antibody titer of Mouse No. 1 is the best, and the optimal titer is 1:13500; therefore, Mouse No. 1 is selected for subsequent experiments.

[0108] (3) Preparation of hybridoma cells

[0109] Resuscitate SP2 / 0 myeloma cells; decapitate Mouse No. 1 and take out the spleen to make a spleen cell suspension; induce cell fusion of myeloma cells and mouse spleen cells by PEG method; after induction of fusion, culture in a 96-well plate using HAT medium, and the culture conditions are 37 °C and 5% CO2; perform semi-medium change culture on the third day and full-medium change culture on the sixth day.

[0110] (4) Screening of hybridoma cells

[0111] On the 7th day of culture, perform positive screening according to the steps in "Detection and Screening of Mouse Serum Titer", and save the hybridoma cells with high titer screened out for standby.

[0112] (5) Induction in animals

[0113] Select BALB / c mice, inject 1 mL of sterilized paraffin oil intraperitoneally, and raise them normally for 7 days; inject the screened hybridoma cells into the mouse peritoneal cavity at a dose of 10 6 cells / mouse; after 7 days, ascites is significantly produced in the mice. Draw the ascites, centrifuge at 8000 r / min for 12 min, and take the supernatant for standby.

[0114] (6) Antibody purification

[0115] Purify the antibody using the saturated ammonium sulfate salting-out precipitation method, measure the antibody concentration after purification, and save it for standby.

[0116] 2. Preparation of CBD monoclonal antibody

[0117] The preparation method is the same as that of CBG monoclonal antibody.

[0118] 3. Preparation of THC monoclonal antibody

[0119] The preparation method is the same as that of CBG monoclonal antibody.

[0120] Example 3

[0121] Preparation of colloidal gold test strip

[0122] (1) Nanogold-labeled antibody: After adjusting the pH value of 100 mL of nanogold solution to 9, mix it with 5 mL of 0.3 mg / mL CBG monoclonal antibody solution, CBD monoclonal antibody solution or THC monoclonal antibody solution, shake for 30 min, add 50 mL of 5% BSA solution for blocking, then centrifuge, discard the supernatant, and resuspend with 10 mL of 20 mM PBS solution to obtain the antibody suspension;

[0123] (2) Detection line preparation: Coat the antibody suspension on one end of the nitrocellulose membrane close to the sample pad to obtain the detection line;

[0124] (3) Quality control line preparation: Coat the goat anti-mouse antibody at 0.4 mg / mL on one end of the nitrocellulose membrane close to the absorbent pad to obtain the quality control line.

[0125] (4) Test strip assembly: Stick the sample pad and the absorbent pad on both ends of the bottom plate, stick the nitrocellulose membrane in the middle of the bottom plate and overlap it with the sample pad and the absorbent pad. After assembly, the colloidal gold test strip is obtained;

[0126] According to the different types and quantities of antibodies, they are prepared respectively according to the above method:

[0127] Single detection line test strips (as Figure 1 shown): CBG colloidal gold test strip, CBD colloidal gold test strip, THC colloidal gold test strip;

[0128] Double detection line test strips: CBG-CBD colloidal gold test strip, CBG-THC colloidal gold test strip, CBD-THC colloidal gold test strip;

[0129] Triple detection line test strip: CBG-CBD-THC colloidal gold test strip.

[0130] Example 4

[0131] Preparation of colloidal gold test strip

[0132] (1) Nanogold-labeled antibody: After adjusting the pH value of 100 mL of nanogold solution to 9, add 1 g of diglycine and 0.5 g of ethanolamine hydrochloride. After mixing evenly, mix it with 5 mL of 0.3 mg / mL CBG monoclonal antibody solution, CBD monoclonal antibody solution or THC monoclonal antibody solution and shake for 30 min. After adding 50 mL of 5% BSA solution for blocking and centrifuging, remove the supernatant, and add 10 mL of 20 mM PBS solution to resuspend to obtain the antibody suspension;

[0133] (2) Detection line preparation: The same as Example 3;

[0134] (3) Quality control line preparation: The same as Example 3;

[0135] (4) Test strip assembly: The same as Example 3;

[0136] According to the different types and quantities of antibodies, they are prepared respectively according to the above method:

[0137] Single detection line test strips: CBG colloidal gold test strip, CBD colloidal gold test strip, THC colloidal gold test strip;

[0138] Dual-detection line test strips: CBG-CBD colloidal gold test strip, CBG-THC colloidal gold test strip, CBD-THC colloidal gold test strip;

[0139] Triple-detection line test strip: CBG-CBD-THC colloidal gold test strip.

[0140] Experimental Example 1

[0141] Detection of monoclonal antibody

[0142] 1. Determination of the coupling ratio of complete antigen

[0143] The conjugated complete antigen was scanned using ultraviolet spectrophotometry, and the amount of hapten conjugated to KLH was calculated according to the additivity principle of absorbance. The calculation formula is as follows:

[0144] Coupling ratio M = (M 抗原 -M KLH ) / M 半抗原 ;

[0145] After calculation, the coupling ratio of CBD to KLH is 1917.6, or the coupling ratio of CBG to KLH is 1820.4, and the coupling ratio of THC to KLH is 1722.8. It shows that the hapten is successfully conjugated to KLH and the coupling ratio is appropriate, which can be used for further experiments.

[0146] 2. Determination of antibody affinity

[0147] Take THC, CBD or CBG respectively and prepare them into 1 μg / mL using coating buffer for coating. The coating method is the same as that in "Detection and screening of mouse serum titer" in Example 2; then perform blocking, and the blocking method is the same as that in "Detection and screening of mouse serum titer" in Example 2; take the monoclonal antibody prepared in Example 2 and dilute it to 5 μg / mL using buffer; dilute the THC-KLH, CBD-KLH or CBG-KLH prepared in Example 1 in a serial dilution; mix the monoclonal antibody with the corresponding THC-KLH, CBD-KLH or CBG-KLH, and incubate in a 37°C incubator for 1 h; then perform secondary antibody, color development, and termination, and the method is the same as that in "Detection and screening of mouse serum titer" in Example 2; calculate the affinity constant K of the monoclonal antibody after measuring the OD value:

[0148] 1 + K / total antigen amount = OD 无抗原 / (OD 无抗原 -OD 不同浓度抗原 );

[0149] After measurement, the affinity constant of the CBG monoclonal antibody prepared in Example 2 is 5.74×10 10 M -1 , and the affinity constant of the CBD monoclonal antibody is 5.47×10 10 M-1 , the affinity constant of the THC monoclonal antibody is 5.59×10 10 M -1 ; the measured affinity constant meets the requirements for preparing the colloidal gold test strip.

[0150] 3. Cross-reactivity determination

[0151] Use the detection method in "Mouse Serum Titer Detection and Screening" in Example 2 to determine whether there is cross-reactivity between the CBG monoclonal antibody, CBD monoclonal antibody, and THC monoclonal antibody, and calculate the cross-reactivity rate:

[0152] Cross-reactivity rate (%) = (IC50 value of the target substance / IC50 value of other substances) × 100%;

[0153] The measurement results are shown in Table 3.

[0154] Table 3 Cross-reactivity rate of monoclonal antibodies in Example 2

[0155] Example 2 CBG monoclonal antibody CBD monoclonal antibody THC monoclonal antibody CBG monoclonal antibody / 0.07 0.08 CBD monoclonal antibody 0.04 / 0.06 THC monoclonal antibody 0.07 0.02 /

[0156] As can be seen from Table 3, the cross-reactivity rate of the monoclonal antibodies prepared in Example 2 is extremely low, and no cross-reactivity will occur.

[0157] Test Example 2

[0158] Colloidal gold test strip detection

[0159] 1. Sensitivity detection

[0160] Prepare CBG, CBD, and THC solutions with concentrations of 1.0 ng / mL, 2.0 ng / mL, 3.0 ng / mL, 4.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, 15.0 ng / mL, 20.0 ng / mL, 25.0 ng / mL, 50.0 ng / mL, 75.0 ng / mL, 100.0 ng / mL, and 150.0 ng / mL respectively as standard solutions, and use the single test line colloidal gold test strips in Examples 3 and 4 for detection; use a dropper to suck the standard solution and drop 3 drops on the sample pad, and observe after standing; when both the test line and the control line show color, it is judged as positive; when the test line does not show color and the control line shows color, it is judged as negative; when only the test line shows color or neither the test line nor the control line shows color, it is judged that the colloidal gold test strip is invalid; for positive samples, record the color development time at the same time.

[0161] The detection results of Example 3 are shown in Table 4. The detection results of Example 4 are similar to those of Example 3, and are not shown here to avoid redundancy.

[0162] Table 4 Sensitivity detection results of the colloidal gold test strip in Example 3

[0163] Standard solution concentration (ng / mL) CBG colloidal gold test strip CBD colloidal gold test strip THC colloidal gold test strip 150 Positive, 65 s Positive, 62 s Positive, 63 s 100 Positive, 63 s Positive, 62 s Positive, 65 s 70 Positive, 64 s Positive, 65 s Positive, 64 s 50 Positive, 68 s Positive, 67 s Positive, 66 s 25 Positive, 69 s Positive, 71 s Positive, 74 s 20 Positive, 75 s Positive, 77 s Positive, 76 s 15 Positive, 77 s Positive, 77 s Positive, 80 s 10 Positive, 81 s Positive, 80 s Positive, 84 s 5 Negative Negative Negative 4 Negative Negative Negative 3 Negative Negative Negative 2 Negative Negative Negative 1 Negative Negative Negative

[0164] As can be seen from Table 4, the minimum detection concentrations of the CBG colloidal gold test strip, CBD colloidal gold test strip, and THC colloidal gold test strip are at least 10 ng / mL, with high sensitivity; in addition, all positive samples can obtain the test results within 85 s, and the test time is short.

[0165] 2. Specificity detection

[0166] Prepare standard solutions of CBG, CBD, THC, caffeine, acetaminophen, and ibuprofen with a concentration of 15.0 ng / mL, and use the colloidal gold test strip in Example 4 for detection; suck the standard solution with a dropper, drop 3 drops on the sample pad, and observe after standing; if the test line and the quality control line show color simultaneously, it is judged as positive; if the test line does not show color and the quality control line shows color, it is judged as negative; if only the test line shows color or neither the test line nor the quality control line shows color, it is judged that the colloidal gold test strip fails.

[0167] The test results of Example 3 are shown in Table 5. The test results of Example 4 are similar to those of Example 3. To avoid redundancy, they are not shown here.

[0168] Table 5 Specificity test results of the colloidal gold test strip in Example 3

[0169]

[0170] As can be seen from Table 5, the colloidal gold test strips prepared in Example 3 can accurately detect CBG, CBD, or THC in the samples, indicating that the colloidal gold test strip of the present invention has good specificity.

[0171] 3. High-temperature resistance detection

[0172] Take out the colloidal gold test strips prepared in Examples 3 and 4 after storing them in an environment of 70 °C for 5, 10, and 15 d for detection. Prepare standard solutions of CBG, CBD, and THC with a concentration of 15.0 ng / mL for detection. The test results of the colloidal gold test strips prepared in Example 4 are shown in Table 6. Since all the colloidal gold test strips prepared in Example 3 failed after being stored at 70 °C for 5 d, the detailed test results of Example 3 are not shown here.

[0173] Table 6 Test results of the colloidal gold test strip in Example 4

[0174]

[0175]

[0176] As can be seen from Table 6, the colloidal gold test strip prepared in Example 4 can still accurately detect after being stored at 70°C for 5 days. However, after being stored at 70°C for 10 days, only the CBD-CBD-THC colloidal gold test strip fails. Compared with the colloidal gold test strip prepared in Example 3, the colloidal gold test strip prepared in Example 4 is more heat-resistant, indicating that the colloidal gold test strip added with diglycine and ethanolamine hydrochloride is more heat-resistant.

[0177] The conventional operations in the operation steps of the present invention are well-known to those skilled in the art and will not be elaborated herein.

[0178] The above-described embodiments have described the technical solutions of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and do not limit the present invention. Any modification, supplement, or substitution in a similar manner within the principle scope of the present invention should be included in the protection scope of the present invention.

Claims

1. A cannabinoid hapten represented by Formula II 2. A cannabinoid complete antigen represented by Formula V 3. A method for preparing the cannabinoid complete antigen according to claim 2, characterized in that, comprising the following steps: (1) Preparation of hapten: Add 4 - 8 parts of cannabinoid molecules, 5 - 10 parts of triethylamine, 6 - 8 parts of DMAP, and 1 - 2 parts of NaOH to 15 - 20 parts of DMF and stir to dissolve. Then add 2 - 3 parts of methyl 4 - chlorobenzoate and heat to 65 - 80 °C and stir for reaction for 2 - 5 h; after the reaction, add 15 - 20 parts of water and extract with ethyl acetate; collect the organic phase, concentrate and dry it, then add 15 - 20 parts of 20% - 30% LiOH solution, and carry out hydrolysis reaction at room temperature for 2 - 5 h; then extract with ethyl acetate, collect the organic phase, adjust the pH to 5 - 6, filter by suction and dry to obtain the cannabinoid hapten described in claim 1; (2) Preparation of complete antigen: Couple the hapten with hemocyanin to obtain the cannabinoid complete antigen described in claim 2; The said cannabinoid molecule is selected from cannabidiol.

4. Use of the hapten described in claim 1 in the preparation of cannabinoid monoclonal antibodies.

5. Use of the complete antigen described in claim 2 in the preparation of cannabinoid monoclonal antibodies.

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