JPWO2016143351A1 - Method for detecting cannabinoids - Google Patents

Abstract

Provided are a method and a kit for simply and accurately determining whether a test sample contains cannabinoids. A plate in which a cannabinoid receptor is immobilized and labeled with a compound having a high affinity for the cannabinoid receptor and a synthetic cannabinoid extracted with a surfactant or an organic solvent are added to react competitively. Thus, it was confirmed that synthetic cannabinoids that are harmful to the human body can be detected easily and accurately without using living cells or special devices.

Description

  The present invention relates to a method for detecting cannabinoids that are main components of dangerous drugs, and a kit for detecting cannabinoids that can be used in such a method.

Cannabis has been used for rituals and treatment since ancient times due to its sedative effect, anti-vomiting effect, euphoria-inducing action, and the like. The components of cannabis include Δ ⁹ -tetrahydrocannabinol ((-)-(6aR, 10aR) -6,6,9-trimethyl-3-pentyl-6a, 7,8,10a -tetrahydro-6H-benzo [c ] chromen-1-ol: Δ9-THC) is a major psychoactive component, and its derivatives include various cannabinoid components including cannabidiol (CBD) and cannabinol (CBN) Has been. Ingestion of cannabis induces catalepsy-like immobility and hypothermia. In addition, it has been reported that abuse can affect the cerebral nervous system because mental dependence is formed or cytotoxicity is induced in a concentration-dependent manner (see, for example, Non-Patent Document 1). There are many countries where the use of is prohibited.

  Cannabis components are also used as pharmaceutical ingredients. For example, Δ9-THC, which is supposed to regulate the mesolimbic dopamine nerve, is regulated by the US Food and Drug Administration (FDA) to control nausea and vomiting associated with chemotherapy, and also suffers from debilitating syndrome. It is approved as a drug for improving the appetite of AIDS patients and is marketed as Marinol (registered trademark) or the like.

  On the other hand, the elucidation of the mechanism of action of cannabinoids via cannabinoid receptors has also progressed rapidly in recent years. For example, the cannabinoid type 1 (CB1) receptor is expressed in large amounts in the nervous system such as the brain and is considered to be involved in the inhibitory control of neurotransmission. In addition, cannabinoid type 2 (CB2) receptors are expressed abundantly in organs and cells of the immune system such as the spleen and tonsils and are considered to be involved in the regulation of inflammatory reactions and immune responses.

  In addition, based on the assumption that in vivo substances that act on the cannabinoid receptors will be present, search for endogenous ligands that bind to the cannabinoid receptors has been conducted. As a result, N-arachidonoylethanolamine (anandamide) was extracted from pig brain as a candidate substance for endogenous ligand (see, for example, Non-Patent Document 2), and palmitoylethanolamine, oleamide, 2-arachidone. In vivo substances that interact with cannabinoid receptors such as glycerol acid (2-AG) have been identified, and these endogenous cannabinoids are analgesic (see, for example, Non-patent Document 3), regulation of feeding (for example, non-patented) It was confirmed to have various physiological activities such as promotion of sleep (see, for example, Reference 4) and sleep promotion (for example, see Non-Patent Document 5) via cannabinoid receptors.

  On the other hand, recently dried plant pieces mixed with a compound with similar pharmacological activity to Δ9-THC are distributed worldwide as so-called “dangerous drugs” as incense and ornamental products. The abuse has become a big social problem. It has been reported that a compound having a stronger action than a natural ingredient is brought about mental dependence at an early stage and is fatal due to its abuse. So far, regulations and enforcement have been implemented for drug components that have been confirmed to be harmful and dangerous, but it has been repeated that new drug components with structures and actions similar to the regulated drug components will appear. And there is a current situation where regulations are not keeping up. For this reason, the Ministry of Health, Labor and Welfare has decided to introduce “Comprehensive Designation” that is subject to regulation if the chemical structure is similar to the “designated drug” regulated by the Pharmaceutical Affairs Law. The compounds having a naphthoylindole skeleton are comprehensively regulated (see, for example, Non-Patent Documents 6 and 7).

  Instrumental analysis such as gas chromatography or liquid chromatography is used in public institutions to identify the so-called dangerous drug action components (see, for example, Non-Patent Document 8). However, it is possible to identify ingredients when there is a standardized drug that is already regulated, but it is extremely difficult to analyze unregulated drugs that do not have a standardized product that continues to increase. is there. Furthermore, it may take several months to synthesize a new standard product, and it often takes time to investigate the cause. As a method for evaluating the toxicity of a drug, an evaluation system using a cultured cell system is widely used as a simple and sensitive method. Synthetic cannabinoids are also observed to be cytotoxic in a concentration-dependent manner (see, for example, Non-Patent Document 9). However, the evaluation method using live cells requires several days for cell culture, and since a cell culture device is required, it can only be carried out by a research institution having a special facility. As described above, since there is no simple inspection method for synthetic cannabinoids, it takes time for the investigation, and development of a new inspection technique is demanded.

  For example, a cannabinoid measurement method in which a functional group having affinity with cannabinoids capable of hydrogen bonding is used as an adsorbent, the cannabinoid in the sample is adsorbed on the adsorbent, the adsorbed cannabinoid is eluted from the adsorbent, and the amount thereof is measured. (For example, refer to Patent Document 1) and methods for determining the presence of a target analyte in a sample using an antibody (for example, refer to Patent Document 2) have been proposed. It is said that it is insufficient from the viewpoint of the general detection of.

JP 2002-062283 A JP 2014-209123 A

Symposium of the 108th Annual Meeting of the Japanese Society of Psychiatry and Neurology, Evaluation of Drug Dependence and Cytotoxicity Science, 258: 1946-1949, 1992 Nature, 394: 277-281, 1998 Nature, 414: 209-212, 2001 Science, 268: 1506-1509, 1995 Promulgation of ministerial ordinance for comprehensive designation of designated drugs, Ministry of Health, Labor and Welfare, February 20, 2013 About partial revision of ministerial ordinance which prescribes use of designated medicine prescribed in Article 2, Paragraph 14 of Pharmaceutical Affairs Law and medical treatment prescribed in Article 76-4 of the same law (enforcement notice) (February 2, 2013) Hyogo Prefectural Institute for Health and Life Sciences Health Science Research Center Report 5: 52-55 Tomiyama, K., et al., Cytotoxicity of synthetic cannabinoids found in “Spice” products: the role of cannabinoid receptors and the caspasecascade in the NG 108-15 cell line. Toxicol Lett, 207: 12-17, 2011

  An object of the present invention is to provide a method and a kit for simply and accurately determining whether or not a test sample contains cannabinoids.

  The present inventors focused on the fact that many of the drug components contained in the dangerous drug exhibit the same action as Δ9-THC, and examined a method for detecting cannabinoids using a cannabinoid receptor. A plate in which a cannabinoid receptor is immobilized is labeled with a compound having a high affinity for the cannabinoid receptor and a synthetic cannabinoid extracted with a surfactant is added and allowed to react competitively. It was confirmed that synthetic cannabinoids that are harmful to the human body can be detected easily and accurately without using cells or special equipment. The present inventors further studied the extraction solvent. First, cannabinoids obtained by diluting synthetic cannabinoids extracted with DMSO (dimethyl sulfoxide) with a buffer containing Tween 20 in order to prevent irreversible denaturation of the receptor, which is a protein. Although the diluted solution and the labeled standard substance reacted competitively, it was difficult to detect cannabinoids. However, when the surfactant is changed from Tween 20 which is a nonionic surfactant to SDS (sodium dodecyl sulfate) which is an anionic surfactant, it is surprisingly possible to detect cannabinoids. I found out. Furthermore, synthetic cannabinoids are extracted using other organic solvents, and then a cannabinoid diluted solution diluted with an anionic surfactant is reacted competitively with a labeled standard substance, thereby accepting a protein receptor. The present invention has been completed by confirming that irreversible denaturation of the protein can be prevented and detection with higher accuracy can be performed.

That is, the present invention is as follows.
[1] A method for detecting cannabinoids in a test sample, comprising the following steps (A) to (E):
(A) a step of preparing a solid phase receptor obtained by solidifying a cannabinoid receptor, a cannabinoid receptor-expressing cell or a cannabinoid receptor-expressing membrane fraction;
(B) a step of preparing a labeled competitive solution containing a labeled standard substance by labeling a standard substance having affinity for the cannabinoid receptor with a labeling substance;
(C) A step of extracting a detection target cannabinoid from a test sample to prepare a sample solution;
(D) The solid phase receptor prepared in step (A) is contacted with the labeled competitive solution and the sample solution, and the labeled standard substance and the cannabinoids to be detected are competitively bound to the cannabinoid receptor, Competitive reaction step to remove unreacted labeled standard;
(E) a measurement step for quantifying the amount of labeled standard substance bound to the cannabinoid receptor;
[2] The detection method according to the above [1], wherein the cannabinoid receptor is a cannabinoid CB1 receptor and / or a cannabinoid CB2 receptor.
[3] The detection method according to the above [1] or [2], wherein the standard substance having affinity for cannabinoid receptors is an agonist having affinity for cannabinoid receptors.
[4] The detection method according to [1] or [2] above, wherein the standard substance having affinity for the cannabinoid receptor is reelamine.
[5] The detection method according to any one of [1] to [4], wherein the labeling substance is peroxidase.
[6] The detection method according to any one of [1] to [5], wherein the test sample is a dangerous drug.
[7] The detection method according to any one of [1] to [6], wherein a sample solution is prepared using an extraction solvent containing a surfactant.
[8] The detection method according to [7] above, wherein the extraction solvent containing a surfactant is an extraction solvent further containing a buffer solution.
[9] The detection method according to [7] or [8] above, wherein the surfactant is SDS.
[10] The detection method according to [9] above, wherein the concentration of SDS is 0.01 to 10%.
[11] The detection method according to [7] or [8] above, wherein the surfactant is Tween20.
[12] The detection method according to the above [11], wherein the concentration of Tween 20 is 0.01 to 10%.
[13] The detection method according to any one of the above [1] to [6], wherein the sample solution is prepared by extraction with an organic solvent and further dilution with a buffer solution containing an anionic surfactant.
[14] The detection method according to the above [13], wherein a sample solution in which the concentration of the organic solvent after dilution with a buffer solution containing an anionic surfactant is 5 to 50% is prepared.
[15] The detection method according to [13] or [14] above, wherein the organic solvent is selected from DMSO, methanol, and ethanol.
[16] The detection method according to the above [15], wherein the organic solvent is DMSO.
[17] The detection method according to any one of [13] to [16], wherein the anionic surfactant is SDS.
[18] The method according to [17] above, wherein the concentration of SDS is 0.01 to 10%.
[19] A label in which a cannabinoid receptor or a cannabinoid receptor-expressing cell or a cannabinoid receptor-expressing membrane fraction is immobilized, and a standard substance having affinity for the cannabinoid receptor is labeled with a labeling substance A cannabinoid detection kit comprising a standard substance and a surfactant for extracting a detection target cannabinoid from a test sample.
[20] A cannabinoid receptor or a cannabinoid receptor-expressing cell or a cannabinoid receptor-expressing membrane fraction immobilized on a solid phase receptor, and a standard substance having an affinity for the cannabinoid receptor labeled with a labeling substance Detection of cannabinoids characterized by comprising a standard substance, an organic solvent for extracting cannabinoids to be detected from a test sample, and an anionic surfactant for diluting the extracted cannabinoids kit.

  According to the present invention, cannabinoids blended in a dangerous drug and having a physiologically or mentally harmful effect can be detected in a wide range regardless of the difference in molecular structure.

It is a graph showing a reverse sigmoid type standard curve. The sample solution containing cannabinoids extracted using SDS at each concentration as the extraction solvent and the labeled standard substance were competitively bound to the cannabinoid receptor, and then the absorbance of the labeled standard substance bound to the cannabinoid receptor was measured. It is a graph to show. (A) PBS containing 0.2% SDS, (b) PBS containing 0.5% SDS, (c) is a graph when PBS containing 1.0% SDS is used. . A sample solution containing cannabinoids extracted using Tween 20 of each concentration as an extraction solvent and a labeled standard substance are competitively bound to a cannabinoid receptor, and then the absorbance of the labeled standard substance bound to the cannabinoid receptor is measured. It is a graph to show. (A) PBS containing 0.2% Tween 20, (b) PBS containing 1.0% Tween 20, (c) PBS containing 5.0% Tween 20, (d) It is a graph at the time of using PBS containing 0% Tween20. The sample solution containing cannabinoids extracted with each concentration of ethanol as an extraction solvent and diluted with 0.2% Tween 20 and the labeled standard substance were competitively bound to the cannabinoid receptor, and then the cannabinoid receptor It is a graph which shows the light absorbency of the labeled standard substance amount couple | bonded. (A) is a sample solution not subjected to ethanol extraction, (b) is a sample solution diluted in 10.0% ethanol, (c) is a sample solution diluted in 20.0% ethanol, and (d) is 40 It is a graph at the time of using the sample liquid diluted to ethanol of 0%. After extraction with DMSO, a sample solution containing cannabinoids in a DMSO solution of 0, 10.0, 20.0, or 40.0% diluted with each concentration of SDS and a labeled standard substance were used as cannabinoid receptors. It is a graph which shows the light absorbency of the labeled standard substance quantity couple | bonded with the cannabinoid receptor after making it couple | bond competitively. (A) is a sample solution diluted with 0.2% SDS, (b) is a sample solution diluted with 0.5% SDS, and (c) is a graph of a sample solution diluted with 10.0% SDS. A sample solution containing cannabinoids in methanol solution of 0, 10.0, 20.0, or 40.0% diluted with 0.5% SDS after extraction with methanol and a labeled standard substance are cannabinoid receptors 2 is a graph showing the absorbance of the amount of labeled standard substance bound to the cannabinoid receptor after competitively binding to. After extraction with ethanol, a sample solution containing cannabinoids in 0, 10.0, 20.0, or 40.0% ethanol diluted with 1.0% SDS and a labeled standard substance were used as cannabinoid receptors. It is a graph which shows the light absorbency of the labeled standard substance quantity couple | bonded with the cannabinoid receptor after making it couple | bond competitively. After extraction with DMSO, a sample solution containing cannabinoids in a solution of 0, 10.0, 20.0, or 40.0% DMSO diluted with each concentration of Tween 20 and a labeled standard substance were used as cannabinoid receptors. It is a graph which shows the light absorbency of the labeled standard substance quantity couple | bonded with the cannabinoid receptor after making it couple | bond competitively. (A) is a sample solution diluted with 0.2% Tween 20, (b) is a sample solution diluted with 0.5% Tween 20, and (c) is a graph of a sample solution diluted with 1.0% Tween 20.

  As a method for detecting cannabinoids in a test sample of the present invention, (A) a cannabinoid receptor-expressing cell or a cannabinoid receptor-expressing membrane fraction, or a solid-phase receptor in which a cannabinoid receptor is immobilized is prepared. (B) a step of preparing a labeled competitive solution containing a labeled standard substance by labeling a standard substance having affinity for the cannabinoid receptor with a labeling substance, and (C) detecting cannabinoids to be detected from the test sample. A step of preparing a sample solution by extraction; (D) bringing the labeled competitive solution and the sample solution into contact with the solid phase receptor prepared in the step (A); Competitive reaction step of removing unreacted labeled standard after competitively binding cannabinoids, (E) Quantifying amount of labeled standard bound to cannabinoid receptor The test sample in the present invention is not particularly limited as long as it is a method comprising a measurement step, and as a test sample in the present invention, blood such as whole blood, serum, plasma and the like, body fluid in a narrow sense such as lymph, spinal fluid, urine, saliva, sweat, In addition to specimens that can be obtained from mammals such as humans such as semen and other body fluids that are secreted and excreted outside the body, nausea, stomach contents, lung lavage fluid, etc. it can.

  The cannabinoids in the present invention include cannabinoids having a naphthoylindole skeleton, cannabinoids having a phenylacetylindole skeleton, cannabinoids having a benzoylindole skeleton, cannabinoids having a cyclohexylphenol skeleton, cannabinoids having a diterpene skeleton, and natural cannabinoids Δ9- In addition to cannabinoids having a dibenzopyran ring similar to THC or a structure similar thereto, analogs and modifications of these compounds, which can include compounds that bind to cannabinoid receptors, The determination of the structure of the compound that exerts an effect of causing a strong mental action such as inducing euphoria, mental dependence, catalepsy-like immobility, and inducing a decrease in body temperature is a feature of the detection according to the present invention. Even a a compound of made is that become unreported, it can be suitably included as cannabinoids. In addition, a pre-metabolic compound that allows a metabolite metabolized in the body to bind to a cannabinoid receptor can also be included in the cannabinoids for convenience as a cannabinoid precursor.

  Examples of cannabinoids having the naphthoylindole skeleton include agonists of CB1 receptor and / or CB2 receptor, JWH-015, JWH-018, JWH-073, JWH-081, JWH-200, JWH-250, JWH. -251, JWH-398 and other compounds of the JWH series synthesized by JWHuffman, and AM-2201, which is a potent agonist of the CB1 receptor and CB2 receptor. Examples of the cannabinoid having the benzoylindole skeleton include AM630, which is a potent CB2 receptor antagonist / inverse agonist. Examples of the cannabinoid having the cyclohexylphenol skeleton include CP55940 that acts on the CB1 receptor and CB2 receptor, and CP47497 that acts on the CB1 receptor. Examples of cannabinoids having a diterpene skeleton include reelamine (1,4a-Dimethyl-7-isopropyl-1,2,3,4,4a, 9,10, which is a potent agonist acting on CB1 receptor and CB2 receptor. , 10a-octahydro-1-phenanthrenemethylamine). Examples of cannabinoids having a structure similar to the dibenzopyran ring include HU-210, which has a very high affinity for Δ9-THC, CB1 receptor and CB2 receptor, and is a potent agonist. Can do.

  The above agonists (agonists), inverse agonists (inverse agonists), and antagonists (antagonists) have the meaning of each term normally used in the art. For example, an agonist is a compound that activates a receptor and induces a physiological effect, an inverse agonist is a compound that stabilizes the receptor in an inactive higher-order structure, and an antagonist is a compound that inhibits the action of an agonist. Explained. Therefore, when the action of the receptor is an action that reduces the function of the cell, the cell function may be increased by inhibiting the action of the antagonist.

  The solid phase receptor prepared in the step (A) is prepared by immobilizing a (purified) cannabinoid receptor or a cannabinoid receptor-expressing cell or a cannabinoid receptor-expressing membrane fraction. A solid phase can be mentioned.

  The cannabinoid receptor is a human CB1 receptor having a binding ability to cannabinoids, a seven-transmembrane, G protein (Gi / Go) -coupled receptor, and comprising a 472 amino acid sequence , And a natural receptor such as human CB2 receptor composed of 360 amino acid sequences (see, for example, Biochemistry, Vol. 83, No. 6, 525-535, 2011), and amino acids of such natural receptor protein A CB1 receptor having one or more amino acids in the sequence substituted, added and / or deleted and / or sugar chain substituted, added and / or deleted, but capable of binding to cannabinoids and / or Or a variant of the CB2 receptor, a partial fragment of the protein of these receptors or variants, or a fusion tamper between the protein of these receptors and another protein A quality, can be exemplified receptor analogs made from proteins capable of binding to cannabinoids. Commercially available purified receptors can also be used, but depending on the purification method, the activity of the receptor may be reduced. Therefore, cannabinoid receptor-expressing cells and receptor-expressing cells such as cannabinoid receptor-expressing membrane fractions may be used. It is desirable to use fractions. In the detection method of the present invention, either the CB1 receptor or the CB2 receptor can be used, but all cannabinoids that bind only to either the CB1 receptor or the CB2 receptor can be detected. In this respect, it is preferable to use CB1 receptor and CB2 receptor.

  Preferred examples of the cannabinoid receptor-expressing cells include cells in which the cannabinoid receptor is expressed as a recombinant protein. Examples of a method for preparing such recombinant receptor protein-expressing cells include a method using a known gene recombination technique. However, by isolating or preparing a DNA encoding the amino acid sequence constituting the cannabinoid receptor, Examples thereof include a method for preparing cells transformed with a vector in which DNA is incorporated into a suitable expression vector and DNA encoding the cannabinoid receptor protein is incorporated.

  The cannabinoid receptor-expressing membrane fraction is not particularly limited as long as it is a cell membrane of a cell expressing a cannabinoid receptor. Examples of such a cell membrane include a cell membrane such as a cell of a postsynaptic neuron and a cell expressing the cannabinoid receptor. Cannabinoid receptor-expressing membrane fraction prepared from

  As a method for preparing such a cannabinoid receptor-expressing membrane fraction from cannabinoid receptor-expressing cells, the medium in which the cannabinoid receptor-expressing cells are grown is removed by suction, the cell surface is washed with a buffer, and then the cells are detached from the medium. Example of adding the medium again to the cells detached from the medium, centrifuging, aspirating the supernatant, suspending in buffer, and resuspending the membrane fraction precipitated by centrifugation with buffer can do. Further, the membrane fraction can be purified by separating the membrane fraction by ultrasonic disruption, physical disruption by friction, ultracentrifugation or the like. After these treatments, a treatment for separating the membrane fraction by ultracentrifugation can be performed in ice-cooling and / or after adding a protease inhibitor. As a buffer for washing the cell surface and suspending the membrane fraction, PBS (-) containing no calcium / magnesium is preferable in that the binding to the dish is loosened and cell detachment is facilitated. Can be listed.

As a method for preparing the solid phase receptor, a cannabinoid receptor suspension is added to the solid phase, or the cannabinoid receptor-expressing cells or the cannabinoid receptor-expressing membrane fraction is converted to 1. 0 × 10 ³ / mL to 1.0 × 10 ⁹ / mL, preferably 1.0 × 10 ⁴ / mL to 1.0 × 10 ⁸ / mL, more preferably 1.0 × 10 ⁵ / mL to 1. Prepare with PBS so as to be equivalent to 0 × 10 ⁷ / mL, add this suspension to the solid phase, 5 minutes to 10 hours, preferably 30 minutes to 5 hours, more preferably 1 hour to 3 hours. An example is a method of blocking by adding PBS containing bovine serum albumin (BSA) after standing and washing with PBS containing Tween 20 (PBST).

  The solid phase used for the preparation of the solid phase receptor includes a plate, a chip, a substrate, a test tube, a porous carrier made of a material capable of forming a solid surface excellent in adhesion of cells and cell fractions, etc. Specifically, polyethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, Polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide , Mention may be made of polysulfone, nitrocellulose membrane, a PVDF film, or the like.

  In the step of preparing the label competitive solution in the step (B), the standard substance having affinity for the cannabinoid receptor includes an agonist having high affinity for the cannabinoid receptor and an inverse having high affinity for the cannabinoid receptor. Agonists and antagonists having high affinity for cannabinoid receptors can be mentioned, and agonists having high affinity for cannabinoid receptors are preferred, and examples thereof include CP-55940, reelamine and oleamide.

  As a method for preparing the labeled standard substance by labeling the standard substance with a labeling substance, the standard substance is a labeled substance such as enzyme, fluorescence, biotin, radioisotope, gold colloid, etc. Examples of the labeling method include labeling, fluorescent labeling, biotin labeling, radioisotope labeling, and colloidal gold labeling. In addition, a commercially available labeling kit or the like can also be used, and specifically, a method of peroxidase labeling a standard substance using a commercially available peroxidase labeling kit can be preferably mentioned. A labeling competition solution can be prepared by suspending these labeling standards in a buffer containing a surfactant as appropriate.

  In the step of preparing the sample solution in the step (C), as an extraction solvent for extracting the detection target cannabinoids from the test sample to prepare the sample solution, a surfactant (method 1) or an organic solvent ( Method 2) can be mentioned.

  In the case of using the surfactant as the extraction solvent in the method 1, it is more preferable to use an extraction solvent further containing a buffer solution. For example, when blood is a test sample, the use amount (volume) of an extraction solvent containing a surfactant and a buffer can be used 2 to 2000 times, preferably 10 to 1000 times that of the test sample.

  As the surfactant in the above method 1, a nonionic surfactant, an anionic surfactant, a cationic surfactant, and an amphoteric surfactant that can prevent irreversible denaturation of a receptor that is a protein are used. Although nonionic surfactant and anionic surfactant are preferable, these can be used 1 type or 2 types or more. Examples of the nonionic surfactant include Tween 20 (polyoxyethylene (20) sorbitan monolaurate), and examples of the anionic surfactant include SDS. Moreover, as a density | concentration of SDS in the method 1, 0.01 to 10% can be mentioned, 0.1 to 5% is preferable, 0.5 to 2% is more preferable, 0.8 to 1.2% is Further preferred. Moreover, as a density | concentration of the said Tween20, 0.01-10% can be mentioned, 0.1-5% is preferable and 1.0-1.2% is more preferable. In the present invention, the concentration (%) represents w / v%.

  The buffer in the above method 1 is not particularly limited as long as it does not interfere with the interaction between the cannabinoid receptor and cannabinoids in the present invention, and includes PBS (Phosphate Buffered Saline), Tris buffer, Examples thereof include citrate buffer and borate buffer.

  When the organic solvent is used as the extraction solvent in the method 2, when the blood is the test sample, the amount of the organic solvent used can be 2 to 2000 times that of the test sample, and 10 to 1000 times. Is preferred. Moreover, the usage-amount of the buffer solution containing an anionic surfactant can be 2-2000 times with respect to the extraction sample extracted with the organic solvent, and 10-1000 times are more preferable.

  The organic solvent in the above method 2 is not particularly limited as long as it can extract the above cannabinoids. Lower alcohol such as methanol, ethanol, isopropanol, N, N-dimethylformamide (DMF), N-methylpyrrolidone ( NMP), aprotic polar solvents such as DMSO, acetonitrile and the like.

  In the above method 2, the concentration of the organic solvent in the sample solution diluted with the buffer solution containing an anionic surfactant is, for example, preferably 5 to 75%, more preferably 10 to 60%, and more preferably 20 to 50%. Is more preferable, and 35 to 45% is particularly preferable.

  More specifically, when the organic solvent is DMSO, the concentration of DMSO in the sample solution diluted with an anionic surfactant is preferably 5 to 75%, more preferably 20 to 60%, and more preferably 30 to 50%. % Is more preferable, and 35 to 45% is particularly preferable.

  When the organic solvent is methanol, the concentration of methanol in the sample liquid diluted with the anionic surfactant is preferably 5 to 75%, more preferably 20 to 60%, still more preferably 30 to 50%, 35 to 45% is particularly preferable.

  When the organic solvent is ethanol, the ethanol concentration in the sample solution diluted with an anionic surfactant is preferably 2 to 50%, more preferably 5 to 15%, and even more preferably 8 to 12%.

  Examples of the anionic surfactant in Method 2 include higher alcohol sulfates, alkyl naphthalene sulfonates, alkyl benzene sulfonates, and alkyl phosphate esters. Specifically, SDS is preferably used. It can be illustrated.

  Examples of the concentration of the anionic surfactant include 0.1 to 1.5%, preferably 0.15 to 1.1%.

  The buffer solution in Method 2 is not particularly limited as long as it does not interfere with the interaction between the cannabinoid receptor and cannabinoids in the present invention, and examples thereof include PBS, Tris buffer solution, citrate buffer solution, borate buffer solution and the like. can do.

  In the competitive reaction step (D), the labeled competitive solution and the sample solution are brought into contact with the solid phase receptor prepared in the step (A), and the labeled standard substance and the cannabinoids are competitively brought into the cannabinoid receptor. As a binding method, the solid phase receptor after blocking is washed with PBST, and the labeled competitive solution and the sample solution are added to and mixed with the solid phase receptor for 5 minutes to 10 hours. , Preferably 30 minutes to 4 hours, more preferably 1 hour to 2 hours, leaving the cannabinoid receptor immobilized on the solid phase receptor to the labeled standard substance in the labeling competition solution, and A method for competitively binding cannabinoids to be detected in the sample solution can be mentioned.

  As a method for removing the unreacted labeled standard substance in the competitive reaction step (D), the solid phase receptor after competitively binding the labeled standard substance and the cannabinoids to be detected with the PBST is used. A method of washing and removing the PBST solution in the solid phase receptor can be mentioned.

  The measurement step of the step (E) is not particularly limited as long as it is a method for quantifying the amount of labeled standard substance bound to the cannabinoid receptor, and after performing a treatment that makes the label detectable with a color former or the like, A method for determining the content of cannabinoids in a test sample using a curve can be mentioned. Specifically, when the amount of cannabinoids to be detected in the test sample is small, since the labeled standard substance binds more to the receptor, the coloration due to the label becomes strong, and the amount of cannabinoids to be detected in the test sample is large Shows a curve of absorbance at which the target cannabinoids decrease in a concentration-dependent manner when such competitive inhibition is established because the labeling standard is captured less by the receptor and the color development due to the label becomes weak. . Therefore, multiple sample solutions with known concentrations of cannabinoids are prepared, a standard curve in which the relationship between absorbance and concentration is clarified is prepared in advance, and the absorbance is obtained from the absorbance obtained when the test sample is processed in the same manner. Obtain the concentration of cannabinoids in the test sample.

  As an ideal form of the standard curve, an inverse sigmoid type standard curve (see, for example, FIG. 1) can be exemplified, and the conditions under which such a standard curve can be created have good reactivity.

  The cannabinoid (s) detected by the method of the present invention is further fractionated by immunoassay such as solid phase enzyme immunoassay (ELISA), affinity chromatography, HPLC, etc., and finally by NMR, LC / MS, etc. More detailed structure can be determined.

  The kit of the present invention includes the above-described cannabinoid receptor or a cannabinoid receptor-expressing cell or a solid phase receptor on which the cannabinoid receptor-expressing membrane fraction is immobilized; a standard substance having affinity for the cannabinoid receptor is labeled A cannabinoid detection kit, a cannabinoid receptor or a cannabinoid receptor-expressing cell, or a cannabinoid receptor thereof, comprising: a labeled standard substance labeled with an activating substance; a surfactant for extracting a cannabinoid to be detected from a test sample; Solid-phase receptor in which the body-expressed membrane fraction is solid-phased; a standard substance that is labeled with a standard substance having affinity for the cannabinoid receptor with a labeling substance; an organic substance for extracting cannabinoids to be detected from a test sample A solvent; an anionic surfactant for diluting the extracted cannabinoids; Can be mentioned example was kit, handling instructions detection method of cannabinoids in the test sample are described are normally attached.

  Hereinafter, the present invention will be described more specifically by way of examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1]
[Examination of Extract 1-PBS in Surfactant]
(Culture of receptor-expressing cells)
Ham F-12 (containing 1 mM L-glutamine) medium (HyClone), fetal bovine serum (FBS) (Hyclone), Zeocin (Invitrogen), G-418 sulfate solution (Wako Pure Chemical Industries, Ltd.) The culture medium added with (made by Co., Ltd.) was used as the preparation medium. As a CB1 receptor-expressing cell, human cannabinoid CB1 receptor aequorin cell line ES-110-A (manufactured by PerkinElmer) is seeded on a 100 mm standard dish to which the above-mentioned preparation medium is added, and 5% CO ₂ at 37 ° C. The cells were cultured for 24-72 hours.

(Preparation of cannabinoid receptor-expressing cell membrane fraction suspension)
The 100 mm dish medium in which the CB1 receptor-expressing cells were sufficiently grown was removed by aspiration, and the cell surface was washed with PBS (-) (manufactured by Wako Pure Chemical Industries, Ltd.), and then 0.05% trypsin / 0.5 mM. 2 mL of EDTA (manufactured by Wako Pure Chemical Industries, Ltd.) was added and allowed to stand at 37 ° C. under 5% CO ₂ , and the cells were detached from the conditioned medium. After adding 10 mL of the above-prepared medium to cells detached from the medium, pipetting collects it in a 50 mL centrifuge tube, and after centrifugation at 1250 rpm for 5 minutes, the supernatant is aspirated and resuspended in PBS (-). The operation of centrifuging was repeated two more times. The cell membrane fraction precipitated by centrifugation was suspended in PBS (−) and stored frozen at −40 ° C. After thawing, the suspension containing the CB1 receptor cell membrane fraction was cultured again with a conditioned medium, and it was confirmed that the cells were not alive (dead cell fraction).

(Preparation of CB1 receptor-expressing cell membrane fraction solid phase microplate)
A suspension containing the CB1 receptor-expressing cell membrane fraction was prepared in PBS so that the cell count was 1.0 × 10 ⁶ / mL, and was added to a Nunc 96-well microplate (manufactured by Thermo Scientific) at 50 μL / Each well was added and allowed to stand at 37 ° C. for 2 hours to immobilize the cell membrane fraction containing the CB1 receptor on the plate. After washing the plate with PBST, PBS containing 1% BSA (manufactured by Sigma) was added at 150 μL / well, and the plate was allowed to stand at 37 ° C. for 1.0 hour for blocking. A body-expressing cell membrane fraction solid phase microplate was prepared.

(Preparation of test sample)
CP-55940 ((−)-cis-3- [2-Hydroxy-4- (), which is known to have a very strong binding ability as a full agonist for both CB1 receptor and CB2 receptor. 1,1-dimethylheptyl) -phenyl] -trans-4- (3-hydroxypropyl) cyclohexanol)) (manufactured by Sigma) was used as a cannabinoid to be detected. Phosphate buffer (PBS: Dulbecco PBS for tissue culture (PBS) containing 0.2%, 1.0, 5.0, or 10.0% Tween 20 (manufactured by MP Biomedicals), which is a buffer containing a surfactant. -), Catalog number 05913, manufactured by Nissui Pharmaceutical Co., Ltd.) and PBS containing 0.2%, 0.5%, or 1.0% sodium dodecyl sulfate (SDS: manufactured by Wako Pure Chemical Industries, Ltd.) Was used as an extraction solvent to prepare a 5000 μg / mL CP-55940 extracted sample.

(Preparation of label competitive solution)
Labeled reelamine as a labeled standard substance was prepared using reelamine (manufactured by Cayman Chemical) as an agonist of CB1 receptor as a standard substance. Using peroxidase as a labeling substance, according to the instructions of Peroxidase Labeling Kit-NH2 (manufactured by Dojindo Laboratories), a labeling kit, enzyme labeling is performed so that the concentration of reelamine is 500 μmol / L, and a peroxidase-labeled reelamine solution Was prepared as a label competition solution.

(Competitive Receptor Bioassay)
The blocked CB1 receptor-expressing cell membrane fraction solid-phase microplate after washing is washed with the PBST, and a labeled reelamine prepared with PBST so as to be a 15 μmol / L peroxidase-labeled reelamine solution is added by 25 μL / well. did. Further, the CP-55940 prepared to 5000 μg / mL was further prepared to 6 μg / mL by a 3-fold dilution stage using PBS containing each surfactant as an extraction solvent, and each sample solution was 25 μL. Per well, and stirred for 1 minute with a plate mixer to mix well the solution in the well. The plate was allowed to stand at 37 ° C. for 1.5 hours and then washed with PBST, and the solution in the well was well removed. As a color developing solution, Sure Blue TMB peroxidase substrate (1-Component) (manufactured by KPL) was added by 100 μL / well and allowed to stand at room temperature under light shielding. While confirming the degree of color development, 100 mol / well of 1 mol / L hydrochloric acid was added appropriately to stop color development. Immediately, the absorbance was measured using a microplate reader under conditions of a main wavelength of 450 nm and a sub wavelength of 620 nm. The results when using PBS containing 0.2%, 0.5%, or 1.0% sodium dodecyl sulfate (SDS: manufactured by Wako Pure Chemical Industries, Ltd.) are shown in FIGS. Each is shown in c). Moreover, the result at the time of using PBS containing 0.2, 1.0, 5.0, or 10.0% Tween20 is shown to Fig.3 (a) (b) (c) and (d), respectively. .

(result)
As is clear from FIGS. 2 (a), (b) and (c), and FIGS. 3 (a), (b), (c) and (d), the absorbance of CP-55940 is obtained when SDS is used as a surfactant. In addition, when Tween 20 was used, the concentration decreased depending on the concentration. This is considered to be because a competitive assay between peroxidase-labeled reelamine, which is a labeled standard substance, and CP-55940, which is a cannabinoid to be detected, has been established. By using this assay system, a synthetic cannabinoid in a dangerous drug is used. It was thought that a kind could be detected. In addition, even when synthetic cannabinoids, which have been thought to be soluble only in organic solvents, are dissolved in a buffer containing a surfactant, the receptor activity is not lost, and the receptor and cannabinoids are bound. It was confirmed that can be measured. In addition, it was shown that stable measurement using a receptor is possible even under a surfactant having a high concentration of 10%, for example, where the antigen-antibody reaction inhibits the reaction.
With regard to the concentration of SDS, 1.0%, which was a reverse sigmoid graph, was considered optimal. Regarding the concentration of Tween 20, it was considered that 1.0% at which the absorbance decreased in a concentration-dependent manner was optimal.

[Comparative Example 1]
[Examination of Extract 2-ethanol in PBS]
(Preparation of CB1 receptor-expressing cell membrane fraction solid phase microplate)
In the same manner as in Example 1, the receptor-expressing cells were cultured, the cannabinoid receptor-expressing cell membrane fraction suspension was prepared, and the CB1 receptor-expressing cell membrane fraction solid phase microplate was prepared.

(Preparation of test sample)
CP-55940 was used as a cannabinoid to be detected. A 50 mg / mL CP-55940 extraction sample was prepared using ethanol (99.5%, reagent grade, catalog number 057-00451, manufactured by Wako Pure Chemical Industries, Ltd.) as an extraction solvent. Such CP-55940 extracted sample was prepared using PBS containing 0.2% Tween 20 as a diluent so that the ethanol concentration would be 10.0, 20.0, or 40.0%. A diluted solution of 5000 ppm CP-55940 was prepared using 0.2% Tween 20 containing 10.0%, 20.0%, 40.0% as diluent.

(Preparation of label competitive solution)
In the same manner as in Example 1 above, a labeled reelamine, which is a labeled standard substance, was prepared using reelamine, which is an agonist of the CB1 receptor, as a standard substance. Using peroxidase as a labeling substance, according to the instructions for Peroxidase Labeling Kit-NH2, reelamine was enzyme-labeled so that the concentration of reelamine was 500 μmol / L, and a peroxidase-labeled reelamine solution was prepared as a labeling competition solution.

(Competitive Receptor Bioassay)
The blocked CB1 receptor-expressing cell membrane fraction solid-phase microplate after washing is washed with the PBST, and a labeled reelamine prepared with PBST so as to be a 15 μmol / L peroxidase-labeled reelamine solution is added by 25 μL / well. did. Furthermore, the diluted solution of CP-55940 (Tween 20) prepared at 5000 ppm was added in an amount of 25 μL / well, and stirred for 1 minute with a plate mixer to mix well the solution in the well. The plate was allowed to stand at 37 ° C. for 1.5 hours and then washed with PBST, and the solution in the well was well removed. As a color developing solution, the above-mentioned Sure Blue TMB peroxidase substrate (1-Component) was added at 100 μL / well and allowed to stand at room temperature under light shielding. While confirming the degree of color development, 100 mol / well of 1 mol / L hydrochloric acid was added appropriately to stop color development. Immediately, the absorbance was measured using a microplate reader under conditions of a main wavelength of 450 nm and a sub wavelength of 620 nm. The results when PBS containing 0%, 10.0%, 20.0%, or 40.0% ethanol and 0.2% Tween 20 are used are shown in FIGS. Shown in c) and (d), respectively.

(result)
As is clear from FIG. 4 (a), when PBS containing 0.2% Tween 20 was used as the extraction solvent, the absorbance decreased when more CP-55940 was present, and inhibition by the competitive reaction was suppressed. As a result, it was shown that a competition assay between peroxidase-labeled reelamine and CP-55940 was established. However, as is clear from FIGS. 4B, 4C and 4D, when 0.2% Tween 20 and ethanol are contained in the extraction solvent, the absorbance increases when more CP-55940 is present. This was considered to be because a non-specific reaction occurred due to denaturation of the receptor under conditions containing ethanol at a high concentration. Therefore, it was confirmed that it was difficult to measure synthetic cannabinoids under the conditions of extracting with Tween 20 and ethanol.

[Example 2]
[Examination of Extract 3-DMSO in PBS]
(Preparation of CB1 receptor-expressing cell membrane fraction solid phase microplate)
In the same manner as in Example 1, the receptor-expressing cells were cultured, the cannabinoid receptor-expressing cell membrane fraction suspension was prepared, and the CB1 receptor-expressing cell membrane fraction solid phase microplate was prepared.

(Preparation of test sample)
CP-55940 was used as a cannabinoid to be detected. Using DMSO (99.0%, reagent grade, catalog number 043-07216, manufactured by Wako Pure Chemical Industries, Ltd.) as an extraction solvent, a 50 mg / mL CP-55940 extraction sample was prepared. Using such CP-55940 extracted sample as a diluent with PBS containing 0.2, 0.5, or 1.0% SDS, the DMSO concentration becomes 10.0, 20.0, or 40.0%. And further diluted to 5000 ppm CP-55940 with 0.2, 0.5, or 1.0% SDS containing 10.0%, 20.0%, or 40.0% DMSO. A solution was prepared.

(Preparation of label competitive solution)
The label competitive solution was prepared in the same procedure as in Example 1 above.

(Competitive Receptor Bioassay)
The blocked CB1 receptor-expressing cell membrane fraction solid-phase microplate after washing is washed with the PBST, and a labeled reelamine prepared with PBST so as to be a 15 μmol / L peroxidase-labeled reelamine solution is added by 25 μL / well. did. Further, the CP-55940 (SDS) diluted solution prepared at 5000 ppm was added in an amount of 25 μL / well, and stirred for 1 minute with a plate mixer to mix well the solution in the well. Thereafter, the absorbance was measured in the same procedure as in Example 1. The results when PBS containing 0.2%, 0.5%, or 1.0% SDS is used as the diluent are shown in FIGS. 5 (a), (b), and (c), respectively.

(result)
As is clear from FIGS. 5 (a), (b) and (c), when DMSO is used as the extraction solvent and then diluted with PBS containing SDS, the absorbance of CP-55940 at any concentration is It was smaller than when DMSO was not used (DMSO 0%). In each graph, assuming that the value of blank (PBS solution) is 100, when diluted with PBS containing 0.2% SDS, 80.4 was shown when DMSO was 0%. When DMSO was 10.0%, it was 71.4, which was 88.81% of the value of 0%. When DMSO was 20.0%, it showed 66.9, which was 83.21% of the value of 0%. When DMSO was 40.0%, it was 52.3, which was 65.05% of the value of 0%. When diluted with PBS containing 0.5% SDS, 74.2 was shown when DMSO was 0%. When DMSO was 10.0%, it was 66.6, which was 89.75% of the value of 0%. When DMSO was 20.0%, it showed 56.8, which was 76.55% of the value of 0%. When DMSO was 40.0%, it was 50.1, which was 67.52% of the value of 0%. When diluted with PBS containing 1% SDS, 53.9 was shown when DMSO was 0%. When DMSO was 10.0%, it was 51.6, which was 95.73% of the value of 0%. When DMSO was 20.0%, it was 48.6, which was 90.17% of the value of 0%. When DMSO was 40.0%, it was 52.0, which was 96.47% of the value of 0%.

  As described above, when synthetic cannabinoids are extracted with DMSO and diluted with PBS containing SDS to prepare a diluted CP-55940 solution, the reactivity is higher than when prepared with only PBS containing SDS. Improved. Specifically, when diluted with 40% DMSO using PBS containing 0.2% SDS, when diluted with 40% DMSO using PBS containing 0.5% SDS, PBS containing 1.0% SDS It was confirmed that the diluted CP-55940 solution was particularly reactive when diluted to 20.0% DMSO.

[Example 3]
[Examination of Extract 4-Methanol in PBS]
(Preparation of CB1 receptor-expressing cell membrane fraction solid phase microplate)
In the same manner as in Example 1, the receptor-expressing cells were cultured, the cannabinoid receptor-expressing cell membrane fraction suspension was prepared, and the CB1 receptor-expressing cell membrane fraction solid phase microplate was prepared.

(Preparation of test sample)
CP-55940 was used as a cannabinoid to be detected. A 50 mg / mL CP-55940 extraction sample was prepared using methanol (99.8%, reagent grade, catalog number 137-01823, manufactured by Wako Pure Chemical Industries, Ltd.) as an extraction solvent. Such a CP-55940 extracted sample was prepared using PBS containing 0.5% SDS as a diluent, so that the methanol concentration was 10.0, 20.0, or 40.0%. 5000 ppm CP-55940 diluted solution was prepared using 0.5% SDS containing 10.0%, 20.0%, or 40.0%.

(Preparation of label competitive solution)
The label competitive solution was prepared in the same procedure as in Example 1 above.

(Competitive Receptor Bioassay)
The blocked CB1 receptor-expressing cell membrane fraction solid-phase microplate after washing is washed with the PBST, and a labeled reelamine prepared with PBST so as to be a 15 μmol / L peroxidase-labeled reelamine solution is added by 25 μL / well. did. Further, the CP-55940 (methanol) diluted solution prepared at 5000 ppm was added at a rate of 25 μL / well, and stirred for 1 minute with a plate mixer, and the solution in the well was mixed well. Thereafter, the absorbance was measured in the same procedure as in Example 1. The results are shown in FIG.

(result)
As is clear from FIG. 6, when methanol was used as the extraction solvent and then diluted with PBS containing 0.5% SDS, the absorbance was smaller than when methanol was not used (methanol 0%). . Specifically, assuming that the value of the blank (PBS solution) is 100, when diluted with PBS containing 0.5% SDS, 74.2 was shown when methanol was 0%. When methanol was 10.0%, it was 68.6, which was 92.45% of the 0% value. When methanol was 20.0%, it was 65.5, which was 88.27% of the value of 0%. When methanol was 40.0%, it was 48.1, which was 64.82% of the value of 0%.

  As described above, when synthetic cannabinoids are extracted with methanol and further diluted with PBS containing SDS to prepare a diluted CP-55940 solution, the reactivity is higher than when prepared with only PBS containing SDS. Improved. Specifically, the CP-55940 diluted solution when diluted to 40% methanol using PBS containing 0.5% SDS had the highest reactivity.

[Example 4]
[Examination of Extract 5-Ethanol in PBS]
(Preparation of CB1 receptor-expressing cell membrane fraction solid phase microplate)
In the same manner as in Example 1, the receptor-expressing cells were cultured, the cannabinoid receptor-expressing cell membrane fraction suspension was prepared, and the CB1 receptor-expressing cell membrane fraction solid phase microplate was prepared.

(Preparation of test sample)
CP-55940 was used as a cannabinoid to be detected. A 50 mg / mL CP-55940 extraction sample was prepared using ethanol as the extraction solvent. Using such CP-55940 extracted sample as a diluent with PBS containing 0.2, 0.5, or 1.0% SDS, the ethanol concentration becomes 10.0, 20.0, or 40.0%. It was prepared as follows. In addition, a 2500 ppm CP-55940 diluted solution was prepared using 0.2%, 0.5, or 1.0% SDS containing 10.0%, 20.0%, 40.0% ethanol as a diluent. did.

(Preparation of label competitive solution)
The label competitive solution was prepared in the same procedure as in Example 1 above.

(Competitive Receptor Bioassay)
The blocked CB1 receptor-expressing cell membrane fraction solid-phase microplate after washing is washed with the PBST, and a labeled reelamine prepared with PBST so as to be a 15 μmol / L peroxidase-labeled reelamine solution is added by 25 μL / well. did. Furthermore, 25 μL / well of the CP-55940 diluted solution prepared to 2500 ppm was added, and the solution in the well was mixed well by stirring for 1 minute with a plate mixer. Thereafter, the absorbance was measured in the same procedure as in Example 1. The results are shown in FIG.

(result)
As is clear from FIG. 7, when ethanol was used as the extraction solvent and then diluted with PBS containing 1.0% SDS, the absorbance was smaller than when ethanol was not used (ethanol 0%). Specifically, assuming that the value of the blank (PBS solution) is 100, when diluted with PBS containing 1.0% SDS, 67.6 was shown when ethanol was 0%. When ethanol was 10.0%, it was 60.7, which was 89.80% of the value of 0%. When ethanol was 20.0%, it showed 63.8, which was 94.38% of the value of 0%. When ethanol was 40.0%, it was 63.1, which was 93.34% of the value of 0%.

  As described above, when synthetic cannabinoids are extracted with ethanol and further diluted with PBS containing SDS to prepare a diluted CP-55940 solution, the reactivity is higher than when prepared with only PBS containing SDS. Improved. Specifically, the reactivity was highest when diluted with 10% ethanol using PBS containing 1.0% SDS.

[Comparative Example 2]
[Examination of Diluent-Tween 20 in PBS]
(Preparation of CB1 receptor-expressing cell membrane fraction solid phase microplate)
In the same manner as in Example 1, the receptor-expressing cells were cultured, the cannabinoid receptor-expressing cell membrane fraction suspension was prepared, and the CB1 receptor-expressing cell membrane fraction solid phase microplate was prepared.

(Preparation of test sample)
CP-55940 was used as a cannabinoid to be detected. CP-55940 was prepared by using 0, 10.0, 20.0, or 40.0% DMSO as an extraction solvent so that CP-55940 was 50 mg / mL. For each such CP-55940 extracted sample, 2500 ppm CP-55940 diluted solution was prepared using PBS containing 0.2, 0.5, or 1.0% Tween 20 as a diluent.

(Preparation of label competitive solution)
The label competitive solution was prepared in the same procedure as in Example 1 above.

(Competitive Receptor Bioassay)
The blocked CB1 receptor-expressing cell membrane fraction solid-phase microplate after washing is washed with the PBST, and a labeled reelamine prepared with PBST so as to be a 15 μmol / L peroxidase-labeled reelamine solution is added by 25 μL / well. did. Furthermore, the diluted solution of CP-55940 (Tween 20) prepared to 2500 ppm was added at a rate of 25 μL / well, and stirred for 1 minute with a plate mixer to mix well the solution in the well. Thereafter, the absorbance was measured in the same procedure as in Example 1. The results when PBS containing 0.2%, 0.5%, or 1.0% Tween 20 is used as a diluent are shown in FIGS. 8 (a), (b), and (c), respectively.

(result)
As is clear from FIGS. 8 (a), (b) and (c), when DMSO is used as an extraction solvent and then diluted with PBS containing Tween 20, the absorbance of CP-55940 at any concentration is It became larger than the case where DMSO was not used. Therefore, it was confirmed that Tween 20 which is a nonionic surfactant is not preferable as a diluted solution after extraction with an organic solvent in the present invention. In addition, since the Tween 20 solution of 0.2 to 1.0% with respect to the value (100) of the blank (PBS) showed a value of 49.9 to 58.2, the measurement itself was appropriately performed. .

  The present invention can detect cannabinoids that are harmful components in dangerous drugs.

Claims (20)

A method for detecting cannabinoids in a test sample, comprising the following steps (A) to (E):
(A) a step of preparing a solid phase receptor obtained by solidifying a cannabinoid receptor, a cannabinoid receptor-expressing cell or a cannabinoid receptor-expressing membrane fraction;
(B) a step of preparing a labeled competitive solution containing a labeled standard substance by labeling a standard substance having affinity for the cannabinoid receptor with a labeling substance;
(C) A step of extracting a detection target cannabinoid from a test sample to prepare a sample solution;
(D) The solid phase receptor prepared in step (A) is contacted with the labeled competitive solution and the sample solution, and the labeled standard substance and the cannabinoids to be detected are competitively bound to the cannabinoid receptor, Competitive reaction step to remove unreacted labeled standard;
(E) a measurement step for quantifying the amount of labeled standard substance bound to the cannabinoid receptor; The detection method according to claim 1, wherein the cannabinoid receptor is a cannabinoid CB1 receptor and / or a cannabinoid CB2 receptor. 3. The detection method according to claim 1, wherein the standard substance having affinity for the cannabinoid receptor is an agonist having affinity for the cannabinoid receptor. 3. The detection method according to claim 1, wherein the standard substance having affinity for the cannabinoid receptor is reelamine. The detection method according to any one of claims 1 to 4, wherein the labeling substance is peroxidase. The detection method according to claim 1, wherein the test sample is a dangerous drug. The detection method according to claim 1, wherein a sample solution is prepared using an extraction solvent containing a surfactant. The detection method according to claim 7, wherein the extraction solvent containing a surfactant is an extraction solvent further containing a buffer solution. The detection method according to claim 7 or 8, wherein the surfactant is SDS. The detection method according to claim 9, wherein the SDS concentration is 0.01 to 10%. The detection method according to claim 7 or 8, wherein the surfactant is Tween20. The detection method according to claim 11, wherein the concentration of Tween 20 is 0.01 to 10%. The detection method according to any one of claims 1 to 6, wherein the sample solution is prepared by further diluting with a buffer containing an anionic surfactant after extraction with an organic solvent. The detection method according to claim 13, wherein a sample solution is prepared in which the concentration of the organic solvent after dilution with a buffer solution containing an anionic surfactant is 5 to 50%. The detection method according to claim 13 or 14, wherein the organic solvent is selected from DMSO, methanol, and ethanol. The detection method according to claim 15, wherein the organic solvent is DMSO. The detection method according to any one of claims 13 to 16, wherein the anionic surfactant is SDS. 18. The method according to claim 17, wherein the concentration of SDS is 0.01 to 10%. A cannabinoid receptor or a cannabinoid receptor-expressing cell or a cannabinoid receptor-expressing membrane fraction immobilized on a solid phase receptor, and a standard substance having an affinity for the cannabinoid receptor labeled with a labeling substance A cannabinoid detection kit comprising a surfactant for extracting a detection target cannabinoid from a test sample. A cannabinoid receptor or a cannabinoid receptor-expressing cell or a cannabinoid receptor-expressing membrane fraction immobilized on a solid phase receptor, and a standard substance having an affinity for the cannabinoid receptor labeled with a labeling substance A cannabinoid detection kit comprising: an organic solvent for extracting a detection target cannabinoid from a test sample; and an anionic surfactant for diluting the extracted cannabinoid.