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A detection method and detection kit for cannabinoid active substances based on cell dopamine release effect

A technology for active substances and detection methods, which is applied in the field of detection methods and detection kits for cannabinoid active substances, can solve the problems of low component content, undetectable, and impossible immunoassay methods, and achieve high-sensitivity detection, Solve the difficulty of supervision and the effect of rapid detection

Active Publication Date: 2022-04-19
浙江诺迦生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, cannabinoids are metabolized quickly in the human body, resulting in extremely low levels of target detection components in the test samples, which cannot be detected
On the other hand, the endless emergence of new synthetic cannabinoids with diverse molecular structures makes antibody-based immunoassays almost impossible, even GC / LC / MS

Method used

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  • A detection method and detection kit for cannabinoid active substances based on cell dopamine release effect
  • A detection method and detection kit for cannabinoid active substances based on cell dopamine release effect
  • A detection method and detection kit for cannabinoid active substances based on cell dopamine release effect

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Construction of pCMV-CB1 plasmid

[0032] Ligase site EcoR I and not 1, the human cannabinoid receptor CB1 gene is cloned under the CMV promoter of the lentiviral expression vector pCDH-CMV-MCS-EF1-Neo, and the eukaryotic expression plasmid pCMV-CB1 (such as figure 1 shown).

Embodiment 2

[0033] Example 2 Establishment of CB1 / SK-N-SH stable cell line and blank control Neo / SK-N-SH stable cell line

[0034] The pCMV-CB1 plasmid, pH1 plasmid, and pH2 plasmid were co-transfected into the lentiviral packaging line cell 293V to prepare CB1 lentivirus, and transfected into SK-N-SH cells. G418 screening and cloning established CB1 / SK-N-SH stable cells. transfected cell lines. Co-transfect the pCDH-CMV-MCS-EF1-Neo plasmid, pH1 plasmid, and pH2 plasmid into the lentiviral packaging line cell 293V to prepare empty vector lentivirus, and transfect SK-N-SH cells, G418 screening, clone establishment blank Control Neo / SK-N-SH stably transfected cell line. Specific steps are as follows:

[0035] 1) Preparation of packaging line cells: One day before transfection, use DMEM-H complete culture medium (containing 10% FBS and 100U / mL penicillin, 100μg / mL streptomycin double antibody) to make lentiviral packaging line cells 293V into 1 ×10 6 Each / mL concentration was inoculated on...

Embodiment 3

[0044] Example 3 Application of the Universal Detection Kit for Cannabinoid Active Substances

[0045] The general detection kit for cannabinoid active substances developed based on the technical solution of the present invention can be applied to the detection of various samples containing cannabinoid active substances. In this embodiment, we take the hair of a marijuana smoker as an example. Specific steps are as follows:

[0046] 1) Hair sample processing: 6 hair samples of marijuana smokers and 8 hair samples of normal people without smoking history were taken, and the numbers are shown in Table 1.

[0047] Cut the hair sample within 3cm of the hair root at 20mg / part, cut it into pieces, put it into a 5mL EP tube, add 2mL of HBSS buffer solution (pH7.4) containing 1% keratinase, add a small amount of zirconium beads and quartz sand, and crush it Shake for 1 minute.

[0048]

[0049] 2) Cell preparation: mix 5×10 cells one day in advance 5 Monoclonal CB1 / SK-N-SH and ...

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Abstract

The invention discloses a detection method of cannabinoid active substances based on cell dopamine release effect and a detection kit thereof. The detection kit includes two components of a detection group kit and a control group kit. The detection group reagent The kit includes a monoclonal cell line CB1 / SK-N-SH stably expressing the human cannabinoid receptor CB1 gene and an ELISA detection kit, and the control kit includes a blank control cell line Neo / SK-N-SH and an ELISA Detection kit. The present invention can realize accurate, highly sensitive and rapid detection of all cannabinoid active substances, whether it is natural cannabinoids or synthetic cannabinoids, including emerging new synthetic cannabinoid active substances, which can solve the difficulty in the supervision of cannabinoid psychoactive substances Problems, fast, efficient, accurate, high-throughput detection of cannabinoid active substances in various samples.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection method of cannabinoid active substances based on cell dopamine release effect and a detection kit thereof. Background technique [0002] The detection and supervision of marijuana drug use by drug users has always been a difficult problem for the regulatory authorities of various countries. Because the current detection methods mainly rely on immunological methods of antibodies (such as anti-tetrahydrocannabinoid antibodies), including chromatography, ELISA, etc.; and large-scale instrumental analysis such as GC-MS and LC-MS. However, cannabinoids are metabolized quickly in the human body, resulting in extremely low levels of target detection components in the test samples, which cannot be detected. On the other hand, new synthetic cannabinoids emerge in an endless stream, and their molecular structures are diverse, making antibody-based immunoassays almost i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867
Inventor 范春雷程向荣
Owner 浙江诺迦生物科技有限公司
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