CN109946451B - Detection method and detection kit for cannabinoids active substances based on concentration change effect of intracellular free calcium ions - Google Patents
Detection method and detection kit for cannabinoids active substances based on concentration change effect of intracellular free calcium ions Download PDFInfo
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Abstract
The invention discloses a detection method of a cannabinoid active substance based on the concentration change effect of intracellular free calcium ions and a detection kit thereof, wherein the detection kit comprises two components, namely a detection group reagent and a negative control group reagent, the detection group reagent comprises a monoclonal cell line CB1/293 stably expressing a humanized cannabinoid receptor CB1 gene and an intracellular free calcium ion probe reagent Fluo 3-AM, and the negative control group reagent comprises a monoclonal cell line CB1/293 stably expressing a humanized cannabinoid receptor CB1 gene, an intracellular free calcium ion probe reagent Fluo 3-AM and a cannabinoid receptor antagonist. The invention can realize accurate and high-sensitivity rapid detection of all cannabinoids active substances, including natural cannabinoids or synthetic cannabinoids, including novel synthetic cannabinoids active substances which are layered endlessly, and can solve the problem of difficult supervision of cannabinoids psychoactive substances.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for detecting a cannabinoids active substance based on a concentration change effect of intracellular free calcium ions and a detection kit thereof.
Background
The detection and supervision of the cannabis drug intake of drug addicts are always difficult problems of supervision departments in various countries. Because the current detection means mainly rely on antibody (e.g., anti-tetrahydrocannabis antibody) immunoassays, including chromatography, ELISA, and the like; and large-scale instrument analysis such as gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. However, cannabinoids metabolize faster in a human body, so that the content of target detection components in a detection sample is extremely low, and the target detection components cannot be detected. On the other hand, the new synthetic cannabinoids are endless and have diverse molecular structures, making antibody-based immunoassays nearly impossible, even GC-MS and LC-MS.
Disclosure of Invention
In view of the above technical problems in the prior art, the present invention aims to provide a method for detecting a cannabinoid active substance based on the effect of change in the concentration of intracellular free calcium ions, and a detection kit thereof.
The conception of the invention is as follows: both natural and a wide variety of synthetic cannabinoids act in the body to act on cannabinoid receptors as targets, thereby producing their biological effects. Cannabinoid receptors include at least two types, CB1 and CB2, CB1 being mainly present in the central nervous system. Cannabinoid receptors belong to the GPCR class, when stimulated by cannabinoid moleculesActivation of signaling pathways following action of mobilizing agents, i.e., Ca stored in endoplasmic reticulum by IP3-DAG signaling pathways2+The cytoplasm is quickly released, the concentration of free calcium ions in cytoplasm is increased, and thus, a cell response is generated; the effect is reversed if a cannabinoid receptor antagonist is used. As a very mature method for measuring the concentration of free calcium ions in cytoplasm, a cell line of stably transformed CB1 is established, and a rapid, efficient, accurate and high-flux universal detection method for cannabinoids active substances based on the change effect of the concentration of the free calcium ions in cells is established.
The detection method of the cannabinoids active substances based on the change effect of the concentration of free calcium ions in the cells is characterized by comprising the following steps:
1) cloning a cannabinoid receptor CB1 gene under a CMV promoter to construct a eukaryotic expression plasmid, transfecting the eukaryotic expression plasmid to a eukaryotic immortalized cell, establishing a CB1 stable cell line, and carrying out culture amplification to obtain a cell culture solution;
2) step 1), adding tetrahydrocannabinol into a cell culture solution to prepare a series of 5 standard solutions with different tetrahydrocannabinol concentrations, and equally dividing each standard solution with the tetrahydrocannabinol concentration into 2 parts of the same solution; wherein 1 part of standard solution is added with an intracellular free calcium ion probe reagent for full reaction, and the reacted standard solution is used for detecting the fluorescence value Y of intracellular free fluorescent calcium ions1(ii) a Adding excessive cannabinoid receptor antagonist into 1 part of the standard solution to obtain negative standard solution, adding intracellular free calcium ion probe reagent into the negative standard solution, reacting, and detecting intracellular free fluorescent calcium ion fluorescence value Y of the reacted negative standard solution2(ii) a By the fluorescence value Y1And fluorescence value Y2The difference is a vertical coordinate, a standard curve is drawn by taking the concentration of the tetrahydrocannabinol as a horizontal coordinate, and a regression equation is calculated;
3) step 1), adding a sample to be detected into a cell culture solution, and preparing to obtain a sample solution; taking 2 parts of the same sample solution, adding an intracellular free calcium ion probe reagent into 1 part of the sample solution, fully reacting, and detecting the intracellular free fluorescent calcium ion fluorescence value Y of the reacted sample solution3(ii) a Adding excessive cannabinoid receptor antagonist into another 1 part of sample solution to obtain negative sample solution, adding intracellular free calcium ion probe reagent into the negative sample solution, reacting, and detecting intracellular free fluorescent calcium ion fluorescence value Y4(ii) a Calculating fluorescence intensity Y3And fluorescence intensity Y4And (3) substituting the difference into the regression equation in the step 2), and calculating the content of the cannabinoid active substances in the sample to be detected.
The method for detecting the cannabinoid active substances based on the change effect of the concentration of free calcium ions in the cells is characterized in that in the step 1), the cannabinoid receptor CB1 gene is a humanized cannabinoid receptor CB1 gene; the eukaryotic expression plasmid is pCMV-CB1 plasmid, and the preparation process comprises the following steps: cloning a humanized cannabinoid receptor CB1 gene to the position under a CMV promoter of a lentivirus expression vector pCDH-CMV-MCS-EF1-Neo, connecting enzyme cutting sites of EcoR I and Not I, and constructing the pCMV-CB1 plasmid.
The method for detecting the cannabinoid active substances based on the change effect of the concentration of the intracellular free calcium ions is characterized in that in the step 1), the process of establishing the CB1 stable cell line comprises the following steps: co-transfecting the pCMV-CB1 plasmid, the pH1 plasmid and the pH2 plasmid to a lentivirus packaging cell 293V to prepare a CMV-CB1 lentivirus; the CMV-CB1 lentivirus is infected into human embryonic kidney cells 293 to obtain 293 cells of stably transformed CMV-CB1, the 293 cells of stably transformed CMV-CB1 are screened by using a conditioned medium containing neomycin G418, and a monoclonal cell line CB1/293 of stably expressing a human cannabinoid receptor CB1 gene is obtained by a picking cloning method.
The method for detecting the cannabinoids active substances based on the change effect of the concentration of free calcium ions in cells is characterized in that in the step 2), tetrahydrocannabinol is added into a cell culture solution to prepare 5 standard solutions with the concentrations of the tetrahydrocannabinol being 0.1, 0.2, 0.4, 0.8 and 1.6ng/mL respectively.
The method for detecting the cannabinoid active substance based on the effect of change of the concentration of free calcium ions in the cells is characterized in that in the step 2), the cannabinoid receptor antagonist is Pyrazoles, Triazoles, Imidazoles or NESS 0327.
The method for detecting the cannabinoids active substances based on the change effect of the concentration of free calcium ions in cells is characterized in that in the step 3), the sample to be detected is urine, blood, hair, dandruff, sweat or saliva of a drug addict, or any one of natural cannabis, natural cannabis products, synthetic cannabis products, sewage, soil and ponds.
The method for detecting the cannabinoids active substance based on the change effect of the concentration of the intracellular free calcium ions is characterized in that the intracellular free calcium ion probe reagents in the step 2) and the step 3) are Fluo 3-AM; in the step 3), the method for detecting the fluorescence intensity of the intracellular free fluorescent calcium ions comprises the following steps: and (3) testing by using a fluorescence microplate reader, selecting 488nm wavelength for excitation, and detecting the fluorescence value of intracellular free fluorescent calcium ions at 526nm wavelength.
The detection kit for the cannabinoid active substances is characterized by comprising two components, namely a detection group reagent and a negative control group reagent, wherein the detection group reagent comprises a monoclonal cell line CB1/293 stably expressing a humanized cannabinoid receptor CB1 gene and an intracellular free calcium ion probe reagent Fluo 3-AM, and the negative control group reagent comprises a monoclonal cell line CB1/293 stably expressing a humanized cannabinoid receptor CB1 gene, an intracellular free calcium ion probe reagent Fluo 3-AM and a cannabinoid receptor antagonist.
Compared with the prior art, the invention has the following beneficial effects:
(1) the limit that an immunoassay method depends on antibodies is avoided, all cannabinoids active substances can be directly and universally detected, the problem of non-specific binding is avoided, and expensive manpower and material resources and a complicated and long-period antibody research and development process are not needed; meanwhile, the limit that the gas chromatography-mass spectrometry and the liquid chromatography-mass spectrometry depend on a reference substance, the limited detection limit and the complex sample pretreatment work are avoided. The technical scheme of the invention can realize accurate and high-sensitivity rapid detection of all cannabinoid active substances, including natural cannabinoids or synthetic cannabinoids, including novel synthetic cannabinoids active substances which are layered endlessly, and can solve the problem of difficult supervision of the cannabinoid psychoactive substances.
(2) Quantitative determination analysis can be carried out by utilizing a mature measuring technology of the concentration of free calcium ions in cytoplasm.
(3) The method can be used for measuring by methods such as a general flow cytometer, a fluorescence microplate reader and the like, and can realize detection in most common laboratories.
(4) Because the detection is based on the biological effect of the receptor signal channel, the result not only can correctly judge whether the cannabinoid active substance exists, but also can quantitatively evaluate the activity of the cannabinoid active substance, and provides reliable experimental basis for supervision and medical intervention.
(5) The invention provides a stable cell line which can respond to all cannabinoids active substances including natural cannabinoids and synthetic cannabinoids through an IP3-DAG signal pathway of CB1, so that the stable cell line can be applied to high-sensitivity universal detection of the cannabinoids active substances, and the stable cell line comprises a CMV (cytomegalovirus) -promoted high-expression gene CB 1; when cannabinoid active substance acts on CB1, IP3-DAG signal path can be activated to store Ca in endoplasmic reticulum2+Rapidly released into cytoplasm and free Ca in cytoplasm2+Can be instantly improved by hundreds of times. Therefore, the mature technology for measuring the concentration of the free calcium ions in cytoplasm can be used for quantitative detection analysis. On the other hand, the invention provides a simple, rapid, efficient, sensitive (picogram-scale), accurate and high-flux universal detection kit for cannabinoids active substances, which is based on an IP3-DAG signal path of a CB1 receptor and realizes rapid, sensitive and accurate detection and quantitative analysis through fluorescence detection for detecting the concentration change of free calcium ions in cytoplasm.
Drawings
FIG. 1 is a plasmid map of pCMV-CB 1;
FIG. 2 is a standard curve plotted on the abscissa of the concentration of the THC standard, minus the ordinate of the fluorescence of the corresponding standard + antagonist set;
FIG. 3 shows the result of the hair sample detection using the universal cannabinoid active substance detection kit of the present invention;
FIG. 4 shows the results of the competitive ELISA method for THC at concentrations of 0-12.8ng/ml, indicating a detection limit for THC >0.4 ng/ml;
FIG. 5 is a standard curve of detection of 0.4-12.8ng/ml THC by competitive ELISA;
FIG. 6 shows the results of the competitive ELISA method for hair samples.
Detailed Description
The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.
In the following examples, the lentiviral packaging line cell 293V was purchased from Biotech, Inc., of Mitsuga, Beijing.
Example 1 construction of pCMV-CB1 plasmid
The humanized cannabinoid receptor CB1 gene was cloned under the CMV promoter of lentiviral expression vector pCDH-CMV-MCS-EF1-Neo by ligating the restriction enzyme sites EcoR I and Not I, and eukaryotic expression plasmid pCMV-CB1 (shown in FIG. 1) was constructed.
EXAMPLE 2 establishment of CB1/293 Stable Trans cell line
The pCMV-CB1 plasmid, the pH1 plasmid and the pH2 plasmid are co-transfected to a lentivirus packaging line cell 293V to prepare CMV-CB1 lentivirus, and are transfected to an HEK293 cell, and a CB1/293 stable transgenic cell line is established by G418 screening and cloning. The method comprises the following specific steps:
1) preparing packaging line cells: one day prior to transfection, the lentiviral packaging line cells 293V were prepared 1X 10 using DMEM-H complete medium (containing 10% FBS and 100U/ml penicillin, 100. mu.g/ml streptomycin double antibody)6One cell culture dish was inoculated at a concentration of 5% CO at 37 ℃ in D19cm2The culture was carried out overnight.
2) Transfection: when the cells in the dish grew to 70% -80% confluence, 20. mu.g of pCMV-CB1 plasmid, 14. mu.g of pH1 plasmid, and 4.7. mu.g of pH2 plasmid were co-transfected into cells 293V in the dish using PEI transfection reagent (see its standard transfection protocol).
3) Collecting viruses: step 2) collecting supernatant after culturing for 72 hours; centrifuging at 4 deg.C and 8000g relative centrifugation force for 15 min, removing precipitate, and filtering the supernatant with 0.45 μm filter membrane to obtain filtered supernatant.
4) And (3) virus concentration: and (3) centrifuging the supernatant virus solution (about 30ml) filtered in the step 3) for 90 minutes at the temperature of 4 ℃ and under the relative centrifugal force of 90000g, removing the supernatant to obtain a precipitate, namely the CMV-CB1 lentivirus, and carrying out resuspension on the precipitate by using 1ml of DMEM-H complete culture solution to obtain the concentrated CMV-CB1 lentivirus.
5) HEK293 cell preparation: 1 is multiplied by 10 in advance5HEK293 cells at an individual/ml concentration were seeded into 1 well of 12 well cell culture plates at 37 ℃ in 5% CO2Cells were cultured to 50% confluence.
6) Transfection of HEK293 cells: sucking out the culture solution for culturing HEK293 cells in the 12-well plate in the step 5), and adding 1ml of CMV-CB1 lentivirus concentrated in the step 4); 37 ℃ and 5% CO2Culturing for 22-24 hr, and replacing with fresh DMEM-H complete culture solution.
7) Screening of CB1/293 stable transfer cells: the HEK293 cells transfected in the step 6) are prepared into 1 × 10 cells after being cultured for more than one week4Inoculating the culture medium into 6-well cell culture plates at the concentration of 2 ml/well; the culture solution is aspirated after the cells adhere to the wall the next day, and 2ml of conditioned medium (DMEM-H complete culture solution containing 600 mug/ml G418) is added; the conditioned medium (DMEM-H complete medium containing 600. mu.g/ml of G418) was replaced 1 time every three days, and the DMEM-H complete medium containing 200. mu.g/ml of G418 was replaced after 21 to 30 days.
8) Establishment of monoclonal CB1/293 stable cell line: the CB1/293 stable transgenic cell prepared in the step 7) is prepared into 1 × 103Inoculating the culture medium into 6-well cell culture plates at the concentration of 2 ml/well; after 10 days of culture, single clones were picked up under a microscope with a 50. mu.l pipette and transferred to a new 6-well cell culture plate for culture, 1 clone/well; after amplification, the CB1 gene is detected by PCR, and the CB1 protein is detected by WB, so that CB1/293 cell clone lines with high, medium and low different expression levels are obtained and are used for detecting cannabinoids active substances with different activity effect degrees.
Example 3 application of Universal detection kit for cannabinoids active substances
The universal detection kit for the cannabinoids active substances, which is developed based on the technical scheme of the invention, can be applied to the detection of various samples containing the cannabinoids active substances. In this example, the hair of a person who sucks hemp is taken as an example. The method comprises the following specific steps:
1) hair sample treatment: hair samples from 6 cannabis smokers and 8 normal persons with no history of smoking were taken and numbered as in table 1.
Clipping hair sample with hair root less than 3cm 20 mg/part, placing into 5ml EP tube after clipping, adding 2ml HBSS buffer solution (pH7.4) containing 1% keratinase, adding small amount of zirconium beads and quartz sand, and vibrating and pulverizing for 1 min on pulverizer to obtain corresponding sample solutions respectively.
2) Cell preparation: 5X 10 in advance one day5Monoclonal CB1/293 cells at an individual/ml concentration were plated in a 96-well cell culture plate at 200. mu.l/well; 37 ℃ and 5% CO2The culture was carried out for 24 hours.
3) Sample adding treatment: the samples were divided into a standard group, a standard + antagonist group, a sample group, and a sample + antagonist group, and the treatment methods were as follows.
Standard substance group: CB1/293 cells are contained in 5 wells, and the final concentration of the THC standard in each well is 0.1, 0.2, 0.4, 0.8 and 1.6ng/ml and 100 mu l/well respectively.
Standard + antagonist group: CB1/293 cells in 5 wells, adding antagonist 0327 NESS into each well, wherein the final concentration of 0327 NESS is 2nM, and incubating for 10min at 37 ℃; the THC standard substance is added into each well, the final concentration of the THC is respectively 0.1, 0.2, 0.4, 0.8 and 1.6ng/ml, and the total concentration is 100 mul/well.
Sample group: CB1/293 cells were added in 42 wells (3 replicate wells each) to the sample solution obtained in step 1), 100. mu.l/well.
Sample + antagonist group: CB1/293 cells with 42 wells (3 duplicate wells each), adding antagonist 0327 NESS, 0327 NESS with final concentration of 2nM and 100 μ l/well into each well, and incubating at 37 deg.C for 10 min; each well was replaced with 100. mu.l/well of a sample solution containing 2nM of 0327 NESS.
4) And (3) detection: for the standard substance group, the standard substance + antagonist group, the sample group and the sample + antagonist treated in the step 3)And the agent groups are used for respectively detecting the fluorescence value of the intracellular fluorescent calcium ions, and the detection method steps of each group are the same. The detection process of the standard group comprises the following steps: adding Fluo 3-AM working solution into the standard substance group treated in the step 3), and culturing for 20 minutes at 37 ℃; adding 3 times of volume of HBSS buffer solution containing 1% FBS, and continuing to culture for 40 minutes; the cells were washed 3 times with HBSS buffer, and centrifuged each time with a 96-well plate to prevent cell loss; culturing at 37 deg.C for 10min, detecting intracellular fluorescent calcium ion with fluorescence microplate reader, exciting at 488nm, detecting at 526nm, and detecting the fluorescence value (labeled as Fluo 3-Ca) of intracellular fluorescent calcium ion at 526nm2+Fluorescence intensity).
5) As a result: taking the concentration of the THC standard substance as an abscissa, subtracting the fluorescence value of the intracellular free fluorescent calcium ions of the corresponding standard substance + antagonist group from the fluorescence value of the intracellular free fluorescent calcium ions of the standard substance group as an ordinate to draw a standard curve, and calculating a regression equation of y = 3176.9x + 1776.3, wherein R is2 = 0.9886, as shown in fig. 2. The result of subtracting the fluorescence value of the corresponding sample + antagonist group from the fluorescence value of the sample group is shown in fig. 3, and the positive sample and the negative sample can be obviously distinguished; calculating according to regression equation for drawing standard curve, wherein no cannabinoid component can be detected in negative samples, and the relative content of cannabinoid in positive samples is (ng/mg): c1: 0.0162, C2:0.0555, C3:0.0355, C4:0.0317, C5:0.0213 and C6: 0.0726.
Comparative example 1: competitive ELISA method for detecting hair sample of hemp sucking person
At present, the cannabis detection mainly depends on immunoassay, including colloidal gold immunochromatography and competitive ELISA. The colloidal gold method is suitable for quick detection, but the ELISA method is 1 order of magnitude higher than the colloidal gold method in sensitivity. The comparative example is the hair sample of hemp suckers detected by ELISA method. The method comprises the following specific steps:
1) hair sample treatment: samples were treated in the same manner as in example 3 to obtain respective sample liquids (hair sample numbers are the same as those in Table 1).
2) Coating antigen: the THC-BSA antigen protein was formulated to a concentration of 1. mu.g/mL with 0.05mol/L carbonate buffer (pH9.6) and coated on a 96-well plate at 100. mu.l/well, and after overnight at 4 ℃, the plate was washed 5 times with PBS buffer (pH7.4) to remove non-specific binding as much as possible.
3) Grouping experiments: the processing method of the standard curve group and the sample group comprises the following steps:
standard curve set: the THC standard substance is prepared into 9 groups with final concentration of 0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8ng/ml by PBS buffer solution (pH7.4), each group contains 5 mu g/ml THC-Mab, and after mixing, 1 hole and 100 mu l/hole of a 96-hole plate coated by THC-BSA are added.
Sample group: adding THC-Mab monoclonal antibody into the sample liquid extracted from the hair, wherein the final concentration of the THC-Mab monoclonal antibody is 5 mu g/ml, uniformly mixing, adding 1 hole of each 96-pore plate coated by THC-BSA, 100 mu l/hole, and 3 multiple holes of each sample.
4) Competitive binding and assay: incubating each group at 37 ℃ for 30 minutes, pouring out the solution, and washing the plate 5 times with PBS (pH7.4); adding a secondary goat anti-mouse antibody labeled with HRP, incubating at 37 ℃ for 30 minutes, pouring out the solution, and washing the plate 5 times with PBS (pH7.4); after the reaction was developed and terminated, the absorbance (OD) was measured at a wavelength of 450nm using a microplate reader.
5) As a result: as shown in FIG. 4, the results indicate the detection limit of THC by the competitive ELISA method>0.4 ng/ml; a standard curve of absorbance (OD) values at THC concentrations of 0.4-12.8ng/ml is shown in FIG. 5, and the regression equation of the standard curve is y = -0.164x + 2.1684, R2= 0.9323. The results of the sample set are shown in fig. 6, only 2 positive samples were detected, and the cannabinoid component was not detected in all the negative samples, as calculated by drawing a standard curve equation, and the tetrahydrocannabinol content in the detected 2 positive samples was (ng/mg): 0.020 part in proportion of C2 and 0.062 part in proportion of C6.
As can be seen from example 3 and comparative example 1, the competitive ELISA method for detecting hair samples of marijuana smokers is far less sensitive and accurate than the system of the present invention. Moreover, ELISA relies on specific antibodies and can only detect a single component, which is ineffective for the infinite variety of synthetic cannabinoids, and the present technology solves this problem well. In addition, the method of the invention is a direct method, and the method has sensitivity, simple and convenient operation, linear range of dose-effect, accuracy of quantification and the like which are obviously superior to the competitive ELISA method.
The statements in this specification merely set forth a list of implementations of the inventive concept and the scope of the present invention should not be construed as limited to the particular forms set forth in the examples.
Claims (5)
1. A method for detecting cannabinoids active substances based on the effect of change of the concentration of free calcium ions in cells is characterized by comprising the following steps:
1) cloning a cannabinoid receptor CB1 gene under a CMV promoter to construct a eukaryotic expression plasmid, transfecting the eukaryotic expression plasmid to a eukaryotic immortalized cell, establishing a CB1 stable cell line, and carrying out culture amplification to obtain a cell culture solution;
2) step 1), adding tetrahydrocannabinol into a cell culture solution to prepare a series of 5 standard solutions with different tetrahydrocannabinol concentrations, and equally dividing each standard solution with the tetrahydrocannabinol concentration into 2 parts of the same solution; wherein 1 part of standard solution is added with an intracellular free calcium ion probe reagent for full reaction, and the reacted standard solution is used for detecting the fluorescence value Y of intracellular free fluorescent calcium ions1(ii) a Adding excessive cannabinoid receptor antagonist into 1 part of the standard solution to obtain negative standard solution, adding intracellular free calcium ion probe reagent into the negative standard solution, reacting, and detecting intracellular free fluorescent calcium ion fluorescence value Y of the reacted negative standard solution2(ii) a By the fluorescence value Y1And fluorescence value Y2The difference is a vertical coordinate, a standard curve is drawn by taking the concentration of the tetrahydrocannabinol as a horizontal coordinate, and a regression equation is calculated;
3) step 1), adding a sample to be detected into a cell culture solution, and preparing to obtain a sample solution; taking 2 parts of the same sample solution, adding an intracellular free calcium ion probe reagent into 1 part of the sample solution, fully reacting, and detecting the intracellular free fluorescent calcium ion fluorescence value Y of the reacted sample solution3(ii) a Adding excessive cannabinoid receptor antagonist into another 1 part of sample solution to obtain negative sample solution, adding intracellular free calcium ion probe reagent into the negative sample solution, reacting, and detecting intracellular free fluorescent calcium ion fluorescence value Y4(ii) a Calculating fluorescence intensity Y3And fluorescence intensity Y4Substituting the difference into the regression equation in the step 2), and calculating the content of the cannabinoid active substances in the sample to be detected;
in the step 1), the cannabinoid receptor CB1 gene is a humanized cannabinoid receptor CB1 gene; the eukaryotic expression plasmid is pCMV-CB1 plasmid, and the preparation process comprises the following steps: cloning a humanized cannabinoid receptor CB1 gene to the position under a CMV promoter of a lentiviral expression vector pCDH-CMV-MCS-EF1-Neo, connecting enzyme cutting sites of EcoR I and Not I, and constructing the pCMV-CB1 plasmid;
in the step 1), the process of establishing the CB1 stable transfer cell line comprises the following steps: co-transfecting the pCMV-CB1 plasmid, the pH1 plasmid and the pH2 plasmid to a lentivirus packaging cell 293V to prepare a CMV-CB1 lentivirus; infecting a human embryonic kidney cell 293 with CMV-CB1 lentivirus to obtain a 293 cell of stably transformed CMV-CB1, screening the 293 cell of stably transformed CMV-CB1 by using a conditioned medium containing neomycin G418, and obtaining a monoclonal cell line CB1/293 stably expressing a human cannabinoid receptor CB1 gene by a picking and cloning method;
in step 2), the cannabinoid receptor antagonist is NESS 0327;
the intracellular free calcium ion probe reagents in the step 2) and the step 3) are Fluo 3-AM.
2. The method according to claim 1, wherein 5 standard solutions with the concentrations of thc being 0.1, 0.2, 0.4, 0.8 and 1.6ng/mL are prepared by adding thc to the cell culture solution in the step 2).
3. The method according to claim 1, wherein in step 3), the sample is urine, blood, hair, dandruff, sweat or saliva of a drug user, or any one of natural hemp, natural hemp product, synthetic hemp product, sewage, soil and pond.
4. The method for detecting cannabinoid-like active ingredients according to claim 1, wherein the step 3) comprises a step of detecting fluorescence intensity of intracellular free fluorescent calcium ions, wherein the step comprises: and (3) testing by using a fluorescence microplate reader, selecting 488nm wavelength for excitation, and detecting the fluorescence value of intracellular free fluorescent calcium ions at 526nm wavelength.
5. The detection kit for the cannabinoid active substances is characterized by comprising two components, namely a detection group reagent and a negative control group reagent, wherein the detection group reagent comprises a monoclonal cell line CB1/293 stably expressing a human cannabinoid receptor CB1 gene and an intracellular free calcium ion probe reagent Fluo 3-AM, the negative control group reagent comprises a monoclonal cell line CB1/293 stably expressing a human cannabinoid receptor CB1 gene, an intracellular free calcium ion probe reagent Fluo 3-AM and a cannabinoid receptor antagonist, and the cannabinoid receptor antagonist is NESS 0327;
the preparation process of the monoclonal cell line CB1/293 for stably expressing the humanized cannabinoid receptor CB1 gene comprises the following steps: the cannabinoid receptor CB1 gene is a human cannabinoid receptor CB1 gene, the human cannabinoid receptor CB1 gene is cloned under a CMV promoter of a lentivirus expression vector pCDH-CMV-MCS-EF1-Neo, the connection enzyme cutting sites are EcoR I and Not I, a pCMV-CB1 plasmid is constructed, then the pCMV-CB1 plasmid, the pH1 plasmid and the pH2 plasmid are co-transfected to a lentivirus packaging cell 293V, and the CMV-CB1 lentivirus is prepared; infecting human embryonic kidney cells 293 with CMV-CB1 lentivirus to obtain 293 cells stably transformed with CMV-CB1, screening the 293 cells stably transformed with CMV-CB1 by using a conditioned medium containing neomycin G418, and obtaining the monoclonal cell line CB1/293 stably expressing the human cannabinoid receptor CB1 gene by a method of selecting clones.
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| WO2002004665A2 (en) * | 2000-07-08 | 2002-01-17 | Aventis Pharma Deutschland Gmbh | Method with a wide range of applications, for identifying modulators of g-protein-coupled receptors |
| WO2004074844A1 (en) * | 2003-02-18 | 2004-09-02 | Astrazeneca Ab | Screening assays for cannabinoid-ligand-type modulators of gpr55 |
| WO2007051063A2 (en) * | 2005-10-28 | 2007-05-03 | Multispan, Inc. | Gpcr expressing cell lines and antibodies |
| WO2019060316A1 (en) * | 2017-09-19 | 2019-03-28 | Cannametrix, Llc | Cell-based assay for quantifying the potency and efficacy of cannabinoids and/or terpenoids, and methods of use thereof |
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