CN114839367A - Preparation and application of cannabinoid monoclonal antibody - Google Patents
Preparation and application of cannabinoid monoclonal antibody Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a cannabinoid monoclonal antibody, belonging to the technical field of biology. The preparation method of the cannabinoid monoclonal antibody comprises the following steps: (1) hapten preparation: reacting cannabinoid molecules with methyl 4-chlorobenzoate to obtain hapten; (2) complete antigen preparation: coupling the hapten and hemocyanin to obtain the complete antigen. The invention further prepares the corresponding monoclonal antibody by preparing the complete antigen of tetrahydrocannabinol, cannabigerol or cannabidiol, and then uses the prepared monoclonal antibody to prepare the colloidal gold test paper. The prepared colloidal gold test paper has strong specificity and high sensitivity. The complete antigen is obtained by coupling tetrahydrocannabinol, cannabigerol or cannabidiol molecules with hemocyanin.
Description
Technical Field
The invention relates to the technical field of biology, in particular to preparation and application of a cannabinoid monoclonal antibody.
Background
Cannabis is an ancient cultivated plant with medicinal development value, and cannabinoids are a general term for a phenolic derivative extracted from cannabis plant, and mainly contain Tetrahydrocannabinol (THC), Cannabigerol (CBG), Cannabidiol (CBD), tetrahydrocannabinolic acid, etc.
CBD is a monomer extracted from cannabis sativa, is the first cannabinoids approved by FDA, is a non-addictive component in cannabis sativa, and has pharmacological effects of anti-spasm, anti-anxiety, anti-inflammatory, anti-oxidation, anti-rheumatism, anti-tumor, etc.
CBG is a psychoactive cannabinoid that has been shown to be very active in protecting brain nerve cells. Researchers at the department of biochemistry and molecular biology, university of madrid compton, spain 2015 found that CBG had neuroprotective properties. CBG can improve motor defect, reserve neuron, improve antioxidant defense level, and has positive effect on neurodegenerative diseases such as multiple sclerosis and Huntington chorea.
THC is the major psychoactive substance in cannabis and is listed in the first category of psychiatric drug specie catalogue. As THC threatens human health. The CBG and the CBD have beneficial pharmacological effects, the rapid detection of various components of the cannabis sativa plant is a key technology for distinguishing high-quality industrial cannabis sativa from drug cannabis sativa, and the rapid detection of THC can meet the requirement of drug detection.
The primary screening methods commonly used for detecting whether a sample contains CBD, CBG or THC include radioimmunoassay, enzyme-linked immunoassay, colloidal Gold Immunochromatography (GICA) and the like. The confirmation method mainly comprises physicochemical detection methods such as High Pressure Liquid Chromatography (HPLC) and gas chromatography-mass spectrometry (GC/MS). Both HPLC and GC/MS tests must be done in the laboratory, require equipment and are expensive to operate by a skilled technician. Radioimmunoassay methods have high sensitivity and specificity, but the instruments are expensive, so that they cannot be popularized and applied. The enzyme-linked immunoassay method retains the advantages of the radioimmunoassay in terms of sensitivity and specificity, but is complicated and time-consuming to operate, and requires a professional technician to operate. The colloidal gold immunochromatography method has the advantages of simplicity, convenience, rapidness, definite result, no need of complex operation skills and special equipment, high sensitivity and specificity and the like, is very suitable for on-site screening of large batches of samples, and becomes a new direction for development of the field of clinical diagnosis.
The field urgently needs to develop a simple, rapid, accurate, reliable, sensitive and cheap detection method suitable for agricultural planting detection, industrial hemp product detection and hemp drug detection. Chinese patent CN101580544A discloses a colloidal gold labeled cannabis sativa and tetrahydrocannabinol monoclonal antibody immunoassay plate, which has the advantages of being capable of rapidly, simply and sensitively detecting tetrahydrocannabinol, but the patent does not make further research and test on other cannabinoid molecules.
Disclosure of Invention
The first purpose of the invention is to provide a cannabinoid complete antigen and a preparation method thereof, and in order to achieve the purpose, the following technical scheme is adopted:
a cannabinoid hapten of formula I or formula II or formula III,
a cannabinoid complete antigen of formula IV or formula V or formula VI,
A method of producing the cannabinoid complete antigen described above, comprising the steps of:
(1) hapten preparation: reacting cannabinoid molecules with methyl 4-chlorobenzoate to obtain hapten;
(2) complete antigen preparation: coupling hapten and hemocyanin (KLH) to obtain complete antigen.
The immunogenicity of the cannabinoid molecules is weaker, and the cannabinoid molecules can form an antigen with stronger immunogenicity after being coupled with a carrier protein; and because the THC, CBG or CBD molecule surface has no active group which can be directly coupled with protein, before coupling with carrier protein, derivatization treatment is carried out to prepare hapten and introduce active carboxyl.
Preferably, the specific operation steps for preparing the hapten in the step (1) comprise:
adding cannabinoid molecules and a catalyst into DMF, stirring for dissolving, adding 4-chlorobenzoic acid methyl ester, heating, and stirring for reacting; adding LiOH solution for hydrolysis reaction after reaction; then extracting by using ethyl acetate, collecting an organic phase, filtering by suction, and drying to obtain the hapten.
More preferably, the cannabinoid molecule is selected from Tetrahydrocannabinol (THC), Cannabigerol (CBG) or Cannabidiol (CBD).
More preferably, the catalyst comprises triethylamine, DMAP and NAOH.
More preferably, the weight ratio of triethylamine, DMAP and NaOH is: 5-10:6-8:1-2.
More preferably, the weight ratio of methyl 4-chlorobenzoate to cannabinoid molecules is from 2-3: 4-8.
More preferably, the reaction temperature of the methyl 4-chlorobenzoate with the cannabinoid molecule is in the range of 65-80 ℃.
More preferably, the reaction time of the methyl 4-chlorobenzoate with the cannabinoid molecule is 2-5 h.
More preferably, the concentration of LiOH is 20-30 wt%.
More preferably, the hydrolysis reaction time is 2-5 h.
More preferably, the hydrolysis reaction temperature is room temperature.
Still more preferably, the specific operation steps of the hapten in the step (1) comprise:
adding 4-8 parts of cannabinoid molecules, 5-10 parts of triethylamine, 6-8 parts of DMAP and 1-2 parts of NAOH into 15-20 parts of DMF (dimethyl formamide) by weight, stirring and dissolving, adding 2-3 parts of 4-methyl chlorobenzoate, heating to 65-80 ℃, and stirring and reacting for 2-5 hours; adding 15-20 parts of water after reaction and extracting by using ethyl acetate; collecting an organic phase, concentrating, drying, adding 15-20 parts of 20% -30% LiOH solution, and performing hydrolysis reaction for 2-5h at room temperature; then extracting with ethyl acetate, collecting the organic phase, adjusting pH to 5-6, filtering, and drying to obtain hapten.
Preferably, the specific operation steps of the complete antigen preparation in step (2) comprise:
dissolving hemocyanin in buffer solution to prepare hemocyanin solution; dissolving the hapten in DMF, and adding EDC and NHS; dropwise adding the hapten solution into the hemocyanin solution, and stirring for reaction after the dropwise addition is finished; and dialyzing after reaction to obtain the complete antigen.
More preferably, the buffer is CBS buffer, pH9.5-9.8, concentration 0.05-0.1M.
More preferably, the weight ratio of hemocyanin to buffer is 2-8: 15-30.
More preferably, the weight ratio of hemocyanin to hapten is 2-8: 1-2.
More preferably, the weight ratio of hapten, DMF solution, EDC and NHS is: 1-2:80-100:2-3:1-2.
More preferably, the reaction temperature is room temperature.
More preferably, the reaction time is 10-15 h.
More preferably, the dialysis system is PBS buffer, pH9-10, concentration 0.1-0.5M.
Still more preferably, the specific operation steps of the complete antigen preparation in step (2) include:
dissolving 2-8 parts by weight of hemocyanin in 20-30 parts by weight of CBS buffer solution (pH9.5-9.8, concentration 0.05-0.1M); dissolving 1-2 parts of hapten in 80-100 parts of DMF, and adding 2-3 parts of EDC and 1-2 parts of NHS; dropwise adding the hapten solution into the hemocyanin solution, and stirring and reacting at room temperature for 10-15h after dropwise adding; after the reaction, the complete antigen is obtained by dialysis with PBS buffer (pH9-10, concentration 0.1-0.5M).
The above-described method for preparing a complete antigen is only a preferred preparation method, and in the preparation of a complete antigen of the present invention, ligation may be performed using any of the prior art techniques known in the art.
Preferably, the preparation method of the cannabinoid monoclonal antibody further comprises the following steps:
detecting and screening the serum titer; preparing hybridoma cells; screening hybridoma cells; inducing in the animal body; and (5) purifying the antibody.
The invention also discloses the cannabinoid monoclonal antibody prepared by the preparation method.
The second purpose of the invention is to provide a colloidal gold test paper which can accurately, quickly and sensitively detect whether a sample contains THC, CBD or CBG.
A colloidal gold test strip contains cannabinoid monoclonal antibodies.
Preferably, the cannabinoid comprises THC, CBD or CBG. The colloidal gold test paper in the prior art is mostly used for detecting THC, the colloidal gold test paper prepared by the invention can be used for simultaneously detecting one or more of THC, CBD or CBG, the detection range is wider, and the detection accuracy is favorably improved.
Preferably, the preparation method of the colloidal gold test paper comprises the following steps:
(1) nano gold labeled antibody: mixing and oscillating the nano gold solution and the antibody, adding a confining liquid, sealing, centrifuging, and carrying out heavy suspension to obtain an antibody suspension;
(2) detection line preparation: coating the antibody suspension on one end of a nitrocellulose membrane close to a sample pad to obtain a detection line;
(3) preparing a quality control line: coating the goat anti-mouse antibody on one end of the nitrocellulose membrane close to the water absorption pad to obtain a quality control line;
(4) assembling test paper: and assembling the sample pad, the water absorption pad paste, the bottom plate and the nitrocellulose membrane to obtain the colloidal gold test paper.
More preferably, (4) the test strip assembly comprises: and attaching the sample pad and the water absorption pad to two ends of the bottom plate, attaching the nitrocellulose membrane in the middle of the bottom plate and overlapping the sample pad and the water absorption pad, and finishing assembly to obtain the colloidal gold test paper.
More preferably, the antibody concentration in the antibody solution is 0.2-0.5 mg/mL.
More preferably, there are 1-3 detection lines, being 1 or 3 of the THC, CBD or CBG detection lines. Therefore, the test paper can be used for independently or simultaneously detecting whether the sample contains 1 or more of THC, CBD or CBG, and when 2 or 3 samples are detected simultaneously, the detection accuracy can be improved.
More preferably, (1) the steps of the nanogold-labeled antibody are as follows:
mixing and oscillating the nano gold solution, the antibody, the diglycol and the ethanolamine hydrochloride, adding a confining liquid, sealing, centrifuging, and carrying out heavy suspension to obtain the antibody suspension.
The addition of the diglycine and the ethanolamine hydrochloride does not cause negative influence on the detection function of the test paper, and conversely, the stability of the colloidal gold and the antibody can be improved, so that the test paper can tolerate higher temperature. The test paper is beneficial to avoiding the test paper from losing efficacy due to high temperature.
Preferably, the weight ratio of diglycine to ethanolamine hydrochloride is 1-3: 0.3-0.8.
The invention also discloses the application of the complete antigen in preparing the cannabinoid monoclonal antibody.
The invention also discloses the application of the diglycine and ethanolamine hydrochloride in the high temperature resistance of the colloidal gold test paper.
The invention also discloses the application of the colloidal gold test paper in detecting whether a sample contains cannabinoid molecules.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, the complete antigens of THC, CBD and CBG are prepared, the monoclonal antibodies of THC, CBD and CBG are further prepared, and the prepared monoclonal antibodies are used for preparing the colloidal gold test paper, wherein the prepared colloidal gold test paper has strong specificity and can accurately detect whether the sample contains 1 or more of THC, CBD or CBG; the sensitivity is high, and the lowest detection concentration is at least 10 ng/mL; in addition, the colloidal gold test paper can obtain a detection result within 90s, and the detection speed is high. When the colloidal gold test paper is prepared, diglycine and ethanolamine hydrochloride are also added into the antibody suspension prepared by the nano-gold labeled antibody, and when the nano-gold solution, the diglycine and the ethanolamine hydrochloride are used together, the high temperature resistance of the colloidal gold test paper can be increased, and the colloidal gold test paper can still be accurately detected after being stored at 70 ℃ for 5-10 days.
Drawings
FIG. 1 shows the assembly sequence of the colloidal gold test paper.
Detailed Description
The exemplary embodiments will be described herein in detail, and the embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present disclosure. Rather, they are merely examples of methods consistent with certain aspects of the present disclosure, as detailed in the appended claims.
The experimental procedures in the following examples are, unless otherwise specified, either conventional or according to the manufacturer's recommendations. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
BALB/c mice were purchased from Ciba Biotech limited; SP2/0 myeloma cells were purchased from Wuhan Protech Life technologies, Inc.
Example 1
Preparation of complete antigens
1. Preparation of CBG complete antigen
(1) Preparation of CBG hapten:
adding 500mg of CBG, 500mg of triethylamine, 600mg of DMAP and 200mg of NaOH into 2000mg of DMF, stirring and dissolving, then adding 300mg of 4-chlorobenzoic acid methyl ester, heating to 75 ℃, stirring and reacting for 3 hours; after the reaction, 1500mg of water is added and ethyl acetate is used for extraction, the organic phase is collected and concentrated and dried, 2000mg of 25 percent LiOH solution is added, and hydrolysis reaction is carried out for 3.5 hours at room temperature; then, ethyl acetate is used for extraction, the organic phase is collected, the pH value is adjusted to 5.5, and the CBG hapten is obtained after suction filtration and drying; the prepared hapten is characterized by using a hydrogen spectrum, and the characterization result is shown as follows, so that the CBG hapten shown in the formula I can be 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,CH 2 ),0.95(m,3H).
(2) complete antigen preparation:
dissolving 5mg of hemocyanin in 25mg of CBS buffer solution (pH9.6, concentration 0.1M) to obtain hemocyanin solution; dissolving 2mg of CBG hapten in 100mg of DMF, and adding 3mg of EDC and 2mg of NHS to prepare a CBG hapten solution; dropwise adding the CBG hapten solution into the hemocyanin solution, and stirring and reacting at room temperature for 10 hours after the dropwise adding is finished; after the reaction, the complete antigen CBG-KLH shown in the formula IV is obtained by dialysis with PBS buffer solution (pH9.6, concentration 0.1M).
2. Preparation of CBD complete antigen
(1) Preparation of the CBD hapten:
the preparation method is the same as the preparation of the CBG hapten to prepare the CBD hapten shown in the formula I. Performing nuclear magnetic structural characterization on the CBD hapten shown in the formula II by using a nuclear magnetic resonance spectrometer, wherein the nuclear magnetic identification result is as follows: 1 H NMR(400MHz,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).
(2) complete antigen preparation:
the preparation method is the same as the preparation of the CBG complete antigen, and finally the CBD-KLH shown in the formula V is prepared.
3. Preparation of THC complete antigen
(1) Preparation of THC hapten:
the preparation method is the same as the preparation of the CBG hapten to prepare the THC hapten shown in the formula III. Performing nuclear magnetic structural characterization on the THC hapten shown in the formula III by using a nuclear magnetic resonance spectrometer, wherein the nuclear magnetic identification result is as follows: 1 H NMR(400MHz,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).
(2) complete antigen preparation:
the preparation method is the same as the preparation of the CBG complete antigen, and the THC-KLH shown in the formula VI is finally prepared.
Example 2
Preparation of monoclonal antibodies
1. Preparation of CBG monoclonal antibody
(1) Animal immunization
Taking 8 BALB/c mice, numbering 1-8, mixing CBG-KLH and Freund's adjuvant for immunization, wherein the weight ratio of CBG-KLH to Freund's adjuvant is 1: 1; the immunization is carried out according to the dose of subcutaneous injection of 50 mug/mouse, the boosting immunization is carried out once every 3 weeks, the dose is halved during the boosting immunization, 3 times of immunization are carried out totally, the tail of the mouse is sampled at 7d after each immunization, and the serum titer of the mouse is determined.
(2) Mouse serum titer detection and screening
Wrapping a plate: diluting antigen D-BSA to 1 μ g/mL with PBS coating solution (pH7.4), mixing, adding into 96-well plate, each well with 100 μ L, and refrigerating at 4 deg.C overnight;
and (3) sealing: after coating, removing coating liquid, washing the plate for 3 times, adding 200 mu L of sealing liquid into each hole, and keeping the temperature in a constant temperature box at 37 ℃ for 1 h; taking out the 96-well plate, discarding the internal liquid, and washing the plate for 1 time;
a first antibody: the antiserum was diluted by different fold starting at 1:500, 100. mu.L per well; control 0.01M PBS; a thermostat at 37 ℃ for 1 h;
secondary antibody: taking out 96-well plate, discarding the inner liquid, washing the plate for 3 times, and adding 100 μ L goat anti-mouse-HRP diluted according to a ratio of 1:20000 into each well; a thermostat at 37 ℃ for 1 h;
color development: taking out the enzyme label plate, discarding the inner liquid, washing the plate for 4 times, adding 100 mu L of TMB developing solution into each hole, and keeping the temperature in a constant temperature box at 37 ℃ for 15 min;
and (4) terminating: adding 100 mu L of 1M HCL solution into each hole, and stopping the reaction; reading the sample immediately on a microplate reader at 450nm, and determining the titer of the sample according to the dilution corresponding to the well with the OD value being more than 2.1 times of the set negative control OD value;
the detection results are shown in table 1;
TABLE 13 CBG monoclonal antibody titre test 7 days after immunization
As can be seen from Table 1, the serum antibody titer of mouse No. 1 was optimal, with the optimal titer being 1: 13500; therefore, mouse No. 1 was selected for subsequent experiments.
(3) Hybridoma cell preparation
Resuscitating the SP2/0 myeloma cells; killing a No. 1 mouse by breaking the neck, taking out the spleen to prepare spleen cell suspension; inducing cell fusion of myeloma cells and mouse spleen cells by PEG method; after induction of fusion, the cells were cultured in a 96-well plate using HAT medium under conditions of 37 ℃ and 5% CO 2 (ii) a And performing half liquid change culture on the third day, and performing full liquid change culture on the sixth day.
(4) Hybridoma cell selection
And (4) culturing for 7 days, carrying out positive screening according to the steps in the mouse serum titer detection and screening, and storing the screened hybridoma cells with high titer for later use.
(5) Induction in animal body
Selecting BALB/c mice, injecting 1mL sterile paraffin oil into the abdominal cavity, and normally feeding for 7 d; the selected hybridoma cells were treated as follows 10 6 Dose/dose was injected into the abdominal cavity of mice; after 7 days, the mouse obviously generates ascites, the ascites is extracted, centrifuged at 8000r/min for 12min, and then the supernatant is taken and stored for later use.
(6) Antibody purification
Purifying the antibody by using a saturated ammonium sulfate salting-out precipitation method, measuring the concentration of the antibody after purification, and storing for later use.
2. Preparation of CBD monoclonal antibody
The preparation method is the same as that of the CBG monoclonal antibody.
3. Preparation of THC monoclonal antibody
The preparation method is the same as that of the CBG monoclonal antibody.
Example 3
Preparation of colloidal gold test paper
(1) Nano gold labeled antibody: after the pH value of 100mL of nano-gold solution is adjusted to 9, mixing the nano-gold solution with 5mL of 0.3mg/mL CBG monoclonal antibody solution, CBD monoclonal antibody solution or THC monoclonal antibody solution, oscillating for 30min, adding 50mL of 5% BSA solution, sealing, centrifuging, removing supernatant, adding 10mL of 20mM PBS solution, and carrying out heavy suspension to obtain an antibody suspension;
(2) detection line preparation: coating the antibody suspension on one end of a nitrocellulose membrane close to a sample pad to obtain a detection line;
(3) preparing a quality control line: coating the goat anti-mouse antibody of 0.4mg/mL on one end of the nitrocellulose membrane close to the water absorption pad to obtain the quality control line.
(4) Assembling test paper: attaching a sample pad and a water absorption pad to two ends of a bottom plate, attaching a nitrocellulose membrane in the middle of the bottom plate and overlapping the sample pad and the water absorption pad, and completing assembly to obtain the colloidal gold test paper;
according to the different types and the different quantities of the antibodies, the antibody is prepared according to the method as follows:
single test line paper (as shown in figure 1): CBG colloidal gold test paper, CBD colloidal gold test paper and THC colloidal gold test paper;
double detection line test paper: CBG-CBD colloidal gold test paper, CBG-THC colloidal gold test paper and CBD-THC colloidal gold test paper;
test paper of three detection lines: CBG-CBD-THC colloidal gold test paper.
Example 4
Preparation of colloidal gold test paper
(1) Nano gold labeled antibody: adjusting the pH value of 100mL of nano gold solution to 9, adding 1g of diglycine and 0.5g of ethanolamine hydrochloride, mixing uniformly, mixing with 5mL of 0.3mg/mL CBG monoclonal antibody solution, CBD monoclonal antibody solution or THC monoclonal antibody solution, oscillating for 30min, adding 50mL of 5% BSA solution, sealing, centrifuging, removing supernatant, adding 10mL of 20mM PBS solution, and carrying out resuspension to obtain an antibody suspension;
(2) detection line preparation: the same as in example 3;
(3) preparing a quality control line: the same as in example 3;
(4) assembling test paper: the same as in example 3;
according to the different types and the different quantities of the antibodies, the antibody is prepared according to the method as follows:
single detection line test paper: CBG colloidal gold test paper, CBD colloidal gold test paper and THC colloidal gold test paper;
double detection line test paper: CBG-CBD colloidal gold test paper, CBG-THC colloidal gold test paper and CBD-THC colloidal gold test paper;
test paper of three detection lines: CBG-CBD-THC colloidal gold test paper.
Test example 1
Detection of monoclonal antibodies
1. Complete antigen coupling ratio assay
Scanning the coupled complete antigen by using an ultraviolet spectrophotometry, and calculating the amount of the coupled hapten on the KLH according to the additivity principle of absorbance, wherein the calculation formula is as follows:
coupling ratio M ═ M (M) Antigens -M KLH )/M Haptens ；
The coupling ratio of CBD to KLH was calculated to be 1917.6 or CBG to KLH was calculated to be 1820.4 and THC to KLH was calculated to be 1722.8. Indicating successful conjugation of the hapten to KLH and moderate conjugation ratios for further testing.
2. Antibody affinity assay
THC, CBD or CBG were coated with 1. mu.g/mL coating buffer solution, and the coating method was the same as that of "detection and screening of mouse serum titer" in example 2; then, sealing is carried out, and the sealing method is the same as that of the mouse serum titer detection and screening in the example 2; the monoclonal antibody prepared in example 2 was diluted to 5. mu.g/mL using a buffer; the THC-KLH, CBD-KLH or CBG-KLH prepared in example 1 was diluted by a factor; mixing the monoclonal antibody with corresponding THC-KLH, CBD-KLH or CBG-KLH, and keeping the temperature in a thermostat at 37 ℃ for 1 h; then secondary antibody, color development and termination are carried out, and the method is the same as that of the mouse serum titer detection and screening in the example 2; the affinity constant K of the monoclonal antibody was calculated after determination of the OD value:
1+ K/total antigen ═ OD Antigen free /(OD Antigen free -OD Different concentrations of antigen )；
The CBG monoclonal antibody prepared in example 2 was determined to have an affinity constant of 5.74X 10 10 M -1 The affinity constant of the CBD monoclonal antibody is 5.47 multiplied by 10 10 M -1 The affinity constant of the THC monoclonal antibody is 5.59 multiplied by 10 10 M -1 (ii) a The measured affinity constant meets the requirement of preparing the colloidal gold test paper.
3. Cross reaction assay
The detection method in "detection and screening of serum titer of mouse" in example 2 was used to determine whether there was cross reaction among the CBG monoclonal antibody, CBD monoclonal antibody, THC monoclonal antibody, and the cross reaction rate was calculated:
the cross-reaction rate (%) (target substance IC50 value/other substance IC50 value) × 100%;
the measurement results are shown in Table 3.
TABLE 3 Cross-reactivity of monoclonal antibodies in example 2
| 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 | / |
As is clear from table 3, the monoclonal antibody produced in example 2 had a very low cross-reactivity and did not cross-react.
Test example 2
Colloidal gold test paper detection
1. Sensitivity detection
CBG, CBD and THC solutions with the concentrations of 1.0ng/mL, 2.0ng/mL, 3.0ng/mL, 4.0ng/mL, 5.0ng/mL, 10.0ng/mL, 15.0ng/mL, 20.0ng/mL, 25.0ng/mL, 50.0ng/mL, 75.0ng/mL, 100.0ng/mL and 150.0ng/mL are respectively prepared as standard solutions, and the single detection line colloidal gold test paper in the embodiments 3 and 4 is used for detection; sucking the standard solution by using a rubber-tipped dropper, dripping 3 drops on the sample pad, and standing and observing after dripping; the detection line and the quality control line are simultaneously developed, and the result is judged to be positive; the detection line is not colored, the quality control line is colored, and the detection line is judged to be negative; only the detection line is colored or the detection line and the quality control line are not colored, and the colloidal gold test paper is judged to be invalid; for positive samples, the development time was also recorded.
The results of the test in example 3 are shown in table 4, and the results of the test in example 4 are similar to those in example 3, and are not shown here to avoid redundancy.
Table 4 example 3 colloidal gold test paper sensitivity test results
| Concentration of Standard solution (ng/mL) | CBG colloidal gold test paper | CBD colloidal gold test paper | THC colloidal gold test paper |
| 150 | Positive, 65s | Positive, 62s | Positive, 63s |
| 100 | Positive, 63s | Positive, 62s | Positive, 65s |
| 70 | Positive, 64s | Positive, 65s | Positive, 64s |
| 50 | Positive, 68s | Positive, 67s | Positive, 66s |
| 25 | Positive, 69s | Positive, 71s | Positive, 74s |
| 20 | Positive, 75s | Positive, 77s | Positive, 76s |
| 15 | Positive, 77s | Positive, 77s | Positive, 80s |
| 10 | Positive, 81s | Positive, 80s | Positive, 84s |
| 5 | Negative of | Negative of | Negative of |
| 4 | Negative of | Negative of | Negative of |
| 3 | Negative of | Negative of | Negative of |
| 2 | Negative of | Negative of | Negative of |
| 1 | Negative of | Negative of | Negative of |
As can be seen from Table 4, the minimum detection concentrations of the CBG colloidal gold test paper, the CBD colloidal gold test paper and the THC colloidal gold test paper are at least 10ng/mL, and the sensitivity is high; in addition, all positive samples can obtain detection results within 85s, and the detection time is short.
2. Specificity detection
Preparing standard solutions of CBG, CBD, THC, caffeine, acetaminophen and ibuprofen with the concentration of 15.0ng/mL, and detecting by using the colloidal gold test paper in the embodiment 4; sucking the standard solution by using a rubber-tipped dropper, dripping 3 drops on the sample pad, and standing and observing after dripping; the detection line and the quality control line are simultaneously developed, and the result is judged to be positive; the detection line is not colored, the quality control line is colored, and the detection line is judged to be negative; and judging that the colloidal gold test paper is invalid if only the detection line is colored or the detection line and the quality control line are not colored.
The results of the test in example 3 are shown in table 5, and the results of the test in example 4 are similar to those in example 3, and are not shown here to avoid redundancy.
Table 5 example 3 test results of specificity of colloidal gold test paper
As shown in Table 5, the colloidal gold test paper prepared in example 3 can accurately detect CBG, CBD or THC in the sample, which indicates that the colloidal gold test paper of the present invention has good specificity.
3. High temperature resistance detection
The colloidal gold test strips prepared in examples 3 and 4 were stored at 70 ℃ for 5, 10 and 15 days and then taken out for testing, and standard solutions of CBG, CBD and THC were prepared at a concentration of 15.0ng/mL for testing, and the test results of the colloidal gold test strips prepared in example 4 are shown in table 6, but the colloidal gold test strips prepared in example 3 all failed after being stored at 70 ℃ for 5 days, and the detailed test results of example 3 are not shown.
Table 6 example 4 colloidal gold test paper test results
As can be seen from table 6, the colloidal gold test paper prepared in example 4 can be accurately detected after being stored at 70 ℃ for 5 days, while only the CBD-THC colloidal gold test paper fails after being stored at 70 ℃ for 10 days; the colloidal gold test paper prepared in example 4 was more resistant to high temperature than the colloidal gold test paper prepared in example 3, thereby indicating that the colloidal gold test paper added with glycylglycine and ethanolamine hydrochloride was more resistant to high temperature.
Conventional operations in the operation steps of the present invention are well known to those skilled in the art and will not be described herein.
The embodiments described above are intended to illustrate the technical solutions of the present invention in detail, and it should be understood that the above-mentioned embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention, and any modification, supplement or similar substitution made within the scope of the principles of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A cannabinoid hapten of formula I or formula II or formula III,
2. a cannabinoid complete antigen of formula IV or formula V or formula VI,
3. a process for the preparation of the cannabinoid complete antigen of claim 2, comprising the steps of:
(1) hapten preparation: reacting the cannabinoid molecule with methyl 4-chlorobenzoate to form the cannabinoid hapten of claim 1;
(2) complete antigen preparation: coupling the hapten to hemocyanin to provide the cannabinoid complete antigen of claim 2;
the cannabinoid molecule is selected from tetrahydrocannabinol, cannabigerol or cannabidiol.
4. A cannabinoid monoclonal antibody produced by immunization of an animal with the cannabinoid complete antigen of claim 2.
5. A colloidal gold test strip comprising the cannabinoid monoclonal antibody of claim 4.
6. The method of preparing the colloidal gold test paper of claim 5, comprising the steps of:
(1) nano gold labeled antibody: mixing and oscillating the nano gold solution and the antibody, adding a confining liquid, sealing, centrifuging, and carrying out heavy suspension to obtain an antibody suspension;
(2) detection line preparation: coating the antibody suspension on one end of a nitrocellulose membrane close to a sample pad to obtain a detection line;
(3) preparing a quality control line: coating the goat anti-mouse antibody on one end of the nitrocellulose membrane close to the water absorption pad to obtain a quality control line;
(4) assembling test paper: and assembling the sample pad, the water absorption pad paste, the bottom plate and the nitrocellulose membrane to obtain the colloidal gold test paper.
7. Use of the hapten of claim 1 for the preparation of a cannabinoid monoclonal antibody.
8. Use of the complete antigen of claim 2 in the preparation of a cannabinoid monoclonal antibody.
9. Use of a colloidal gold test strip as defined in claim 5 for detecting the presence of cannabinoid molecules in a sample.
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| CN115160135A (en) * | 2022-08-10 | 2022-10-11 | 郑州左安检测科技有限公司 | Preparation method and application of cannabidiol hapten and complete antigen |
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| CN115160135A (en) * | 2022-08-10 | 2022-10-11 | 郑州左安检测科技有限公司 | Preparation method and application of cannabidiol hapten and complete antigen |
| CN115160135B (en) * | 2022-08-10 | 2023-11-03 | 郑州左安检测科技有限公司 | Preparation method and application of cannabidiol hapten and complete antigen |
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