JPWO2015122484A1 - Cannabis component extraction method, cannabis component inspection device and cannabis component inspection method - Google Patents

Abstract

An object of the present invention is to provide a cannabis extraction method, a cannabis component inspection device, and a cannabis component inspection method in which the use of an alcohol component is restricted. A method for extracting cannabis components, comprising placing a target plant test piece suspected of being cannabis in a solution having an alcohol concentration of 30 wt% or less and extracting the components of the test piece into the solution.

Description

  The present invention relates to a method for extracting cannabis components for inspection of cannabis, which is an illegal drug, an inspection device for use in inspection of cannabis components, and a method for inspecting cannabis components.

  Conventionally, in cannabis inspection, a simple inspection using a color reagent is performed by immersing cannabis leaves and corolla in an organic solvent such as alcohol to extract components. Using a reagent or the like, it is temporarily recognized that the discovered object is cannabis. In that case, use the reagents in the kit.

  In the simple test, a reagent having a characteristic of showing a specific reaction color for a specific drug is used as a reagent. In the case of cannabis, the most widely used is called Duchenoa reagent, and the reagent reacts with THC (tetrahydrocannabinol), which is a component of cannabis, to show a color reaction (Non-patent Document 1). Specifically, after three kinds of liquids are added in order to the cannabis and reacted, after a while, the liquid is divided into two upper and lower layers, and the upper layer exhibits a transparent light purple color and the lower layer exhibits an opaque blue purple color.

  However, the point of determination is that the liquid to which the reagent is added is divided into two upper and lower layers, and the lower layer has a blue-violet color. In reality, it is difficult to determine and accurate determination is not easy.

  As described above, it is not easy to determine the color reaction of the cannabis simple test. For example, an erroneous determination may occur in a dark vehicle. In addition, among the three liquids included in the kit, there is also a powerful medicine, hydrochloric acid, which is very dangerous and requires attention depending on the handling.

  Moreover, in the conventional method, the cannabis component was extracted using a 100% organic solvent (Patent Document 1). Furthermore, there has been a report that it is necessary to separate and purify hallucinatory components in cannabis by repeating operations such as liquid-liquid extraction after extraction using an organic solvent such as methanol (Patent Document 2).

Japanese Patent No. 4435993 JP 2012-69516 A

Marja H. Niemi et al,, JOURNAL OF MOLECULAR BIOLOGY, (2010), VOL400 (4), pages 803-814

  An object of this invention is to provide the method of extracting a cannabis component from a cannabis plant piece using the extraction solution of low alcohol concentration.

  In the field of cannabis control, it is necessary to detect cannabis on-site, and there has been a demand for safe judgment kits and judgment methods with few false judgments and no use of deleterious substances.

  As a safe method for measuring cannabis components that can be easily carried out, have few misjudgments, and do not use deleterious substances, an immunological measurement method using an antibody against cannabis components and utilizing an antigen-antibody reaction is desirable. Thought.

  However, conventionally, an organic solvent such as alcohol is essential for extraction of the cannabis component, and 100% of the organic solvent has been used for the extraction. The antibody has low resistance to the organic solvent required for extraction of the cannabis component, and the cannabis component extracted with the organic solvent could not be measured using the antigen-antibody reaction.

  The present inventors need to measure cannabis components (THC-A, THC and CBN) by immunoassay using antigen-antibody reaction in order to enable measurement of cannabis components using antigen-antibody reaction. The concentration of the cannabis component was examined. According to Marja H. Niemi et al,, JOURNAL OF MOLECULAR BIOLOGY, (2010), VOL400 (4), pages 803-814, the presence of 3 ng / mL cannabis components when using antibodies against cannabis components Can be detected. However, in order to optically detect the fluorescence intensity emitted from the antibody after the antigen-antibody reaction using a fluorescently labeled antibody, the fluorescence intensity when reacted with a cannabis component having a concentration of 1 μg / mL or more is required. Therefore, the concentration of the cannabis component necessary for carrying out the antigen-antibody reaction was set to 1 μg / mL (4 μg / mL was diluted 4-fold), and subsequent studies were conducted. Since the concentration of the cannabis component required for the antigen-antibody reaction is 1 μg / mL or more, the concentration of the cannabis component required for extraction is assumed to be 4 μg / mL or more when it is assumed that the specimen is diluted 4-fold during the antigen-antibody reaction. It is.

  Next, the present inventors prepared extraction solutions in which the content ratio of methanol used for extraction was changed, and measured the amount of cannabis components extracted. Surprisingly, it became clear for the first time that a solution containing 30% by weight or less of methanol can be extracted in an amount exceeding the lower limit of analysis of cannabis components.

  Furthermore, even when an antigen-antibody reaction is directly carried out using an extract containing 30 wt (wt)% or less of methanol as a specimen, the antigen-antibody reaction is not inhibited by methanol. The inventors have found that it can be measured, and have completed the present invention.

That is, the present invention is as follows.
[1] A method for extracting cannabis components, comprising placing a target plant test piece suspected of being cannabis in a solution having an alcohol concentration of 30 wt% or less and extracting the components of the test piece into the solution.
[2] The method for extracting cannabis components according to [1], using a solution containing no alcohol.
[3] The solution is a phosphate buffered saline (PBS-T) containing a surfactant, a phosphate buffered saline (PBS) containing no surfactant, or a pure water solution, [1] or [2] Cannabis component extraction method.
[4] The method for extracting a cannabis component according to any one of [1] to [3], wherein the alcohol is selected from the group consisting of methanol, ethanol, and propanol.
[5] The method for extracting a cannabis component according to any one of [1] to [4], wherein the cannabis component is tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THC-A), or cannabinol (CBN).
[6] The method for extracting a cannabis component according to any one of [1] to [5], wherein the target plant test piece includes any part selected from the group consisting of leaves, stems, roots, seeds, and petals.
[7] A testing device used to detect cannabis components,
A first container and a second container;
The first container contains a solution having an alcohol concentration of 30 wt% or less,
The second container contains an antibody against the cannabis component,
Put the target plant test piece suspected of being cannabis in the first container, extract the components of the test piece into a solution having an alcohol concentration of 30 wt% or less,
A test device for detecting a cannabis component that causes an antigen-antibody reaction in a second container by adding a predetermined amount of the extract in the first container to the second container.
[8] The test device for detecting the cannabis component according to [7], wherein the alcohol final concentration during the antigen-antibody reaction occurring in the second container is 7.5 wt% or less.
[9] The solution is a phosphate buffered saline (PBS-T) containing a surfactant, a phosphate buffered saline (PBS) containing no surfactant, or a pure water solution, [7] or [8] An inspection device for detecting the cannabis component.
[10] The test device for detecting the cannabis component according to any one of [7] to [9], wherein the alcohol is selected from the group consisting of methanol, ethanol, and propanol.
[11] For detecting the cannabis component according to any one of [7] to [10], wherein the cannabis component is tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THC-A), or cannabinol (CBN). Inspection device.
[12] For detecting the cannabis component according to any one of [7] to [11], wherein the target plant test piece includes any part selected from the group consisting of leaves, stems, roots, seeds, and petals Inspection device.
[13] An antibody against a cannabis component comprises an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide, and one or both of the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide Are labeled with a fluorescent dye that is quenched in a state where the antibody heavy chain variable region polypeptide or the antibody light chain variable region polypeptide is labeled, and the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide are antigens. The device for a test for detecting the cannabis component in any one of [7]-[12] which is an antibody by which quenching is canceled and a fluorescence intensity increases when a complex is formed via the.
[14] An antibody against the cannabis component comprises an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide, and one or both of the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide Is labeled with a fluorescent dye, and when the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide bind to a cannabis component as an antigen to form a complex, the antigen and the antigen binding protein The complex becomes a quencher of the fluorescent dye, and the antigen concentration and the fluorescence intensity of the fluorescent dye are negatively correlated, and the antigen and the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide The cannabis according to any one of [7] to [12], which is an antibody whose fluorescence intensity decreases when the fluorescent dye is more strongly quenched when a complex is formed Inspection device for detecting components.
[15] The antibody against the cannabis component is an antibody produced by a hybridoma that produces an antibody that binds to tetrahydrocannabinol (THC) or a derivative thereof deposited internationally under the accession number NITE BP-01970 [7] to [12] A testing device for detecting the cannabis component according to any one of [12].
[16] Place a target plant test piece suspected of being cannabis in a solution having an alcohol concentration of 30 wt% or less, elute the components of the test piece into the solution, and add the extract to an antibody against the cannabis component. A method for testing a cannabis component, comprising causing an antigen-antibody reaction.
[17] The method for testing a cannabis component according to [16], wherein the final alcohol concentration during the antigen-antibody reaction is 7.5 wt% or less.
[18] The solution is a phosphate buffered saline (PBS-T) containing a surfactant, a phosphate buffered saline (PBS) containing no surfactant, or a pure water solution, [16] or [17] The method for testing cannabis components.
[19] The method for inspecting a cannabis component according to any one of [16] to [18], wherein the alcohol is selected from the group consisting of methanol, ethanol, and propanol.
[20] The method for testing a cannabis component according to any one of [16] to [19], wherein the cannabis component is tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THC-A), or cannabinol (CBN).
[21] The test method for cannabis components according to any one of [16] to [20], wherein the target plant test piece includes any part selected from the group consisting of leaves, stems, roots, seeds, and petals.

  This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2014-025914, which is the basis of the priority of the present application.

  The method of the present invention is a method of extracting cannabis components from cannabis plant pieces with a solution having an alcohol concentration of 30 wt% or less, and the cannabis components extracted by the method use an antigen-antibody reaction using an extract containing alcohol as a specimen. It can be detected by an immunological assay. The cannabis component can be detected by the cannabis component extraction method and the inspection method of the present invention. In addition, cannabis components can be extracted and detected without using deleterious substances such as hydrochloric acid. According to the present invention, the cannabis component can be detected on-site simply and safely.

It is a figure which shows the structure of THC, THC-A, and CBN. It is a longitudinal cross-sectional view of the device for a test | inspection which performs extraction of a cannabis component and an antigen antibody reaction. It is a figure which shows the external appearance of a fluorescence tester. It is a figure which shows the internal structure of a fluorescence tester. It is a figure which shows the principle of the antigen identification by the antigen antibody reaction by excitation light irradiation. It is a figure which shows the measurement system of the fluorescence tester shown in FIG. It is a figure which shows the method of performing the extraction of a cannabis component and an antigen antibody reaction using the device for a cannabis component test | inspection shown in FIG. It is a figure which shows the outline | summary of the extraction method of a cannabis component. It is a figure which shows the relationship of the detection of the cannabis component by alcohol concentration and an antigen antibody reaction. It is a figure which shows the relationship of the detection of the cannabis component (THC) by alcohol concentration and an antigen antibody reaction. It is a figure which shows the relationship of the detection of the cannabis component (THC-A) by alcohol concentration and an antigen antibody reaction. It is a figure which shows the reactivity with THCA, THC, and CBN of the monoclonal antibody which hybridoma A-04 produces.

  Hereinafter, the present invention will be described in detail.

  The present invention is a method for extracting a cannabis component for examining whether or not a test object which is a target plant test piece suspected of being cannabis is cannabis.

  Cannabis is also referred to as marijuana, and refers to a product obtained by drying or resinating a liquid of a corolla or leaf of a Asa belonging to the genus Cannabis. In the present invention, a plant part of Asa such as a leaf, stem, root, seed and petal or a plant fragment thereof is used as a specimen. Usually, these plant parts or plant pieces are used as dry cannabis in a dry state. In the present invention, preferred test objects are these dry cannabis. In particular, dried cannabis plant pieces are preferred.

Cannabis contains a chemical substance called cannabinoid as a cannabis component, and cannabinoid has various pharmacological actions such as euphoria, analgesic action and hallucination action. Examples of cannabinoids include tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THC-A), cannabinol (CBN), and cannabidiol (CBD). In the present invention, among these, tetrahydro having a strong hallucinating action. The purpose is to extract cannabinol (THC), tetrahydrocannabinolic acid (THC-A) or cannabinol (CBN). THC has double bond positional isomers, Δ ⁸ -THC and Δ ⁹ -THC. Reference to THC includes Δ ⁸ -THC and Δ ⁹ -THC. The chemical structural formulas of THC, THC-A and CBN are shown in FIG.

  Conventionally, in cannabis inspection, the cannabis components of THC, THC-A, and CBN have been extracted from cannabis plant pieces using an organic solvent such as methanol. For example, it was recognized that it was necessary to use a 100 wt (mass)% high purity organic solvent. On the other hand, since organic solvents inhibit antigen-antibody reactions, it was not possible to directly measure cannabis components extracted with high-concentration organic solvents using antigen-antibody reactions. Therefore, it was recognized that cannabis components in cannabis plant pieces could not be extracted and the cannabis components could be measured directly using an antigen-antibody reaction using the extract.

  In the present invention, a cannabis component is extracted from a cannabis plant piece using a low-concentration organic solvent, which has been conventionally recognized as being unable to extract the cannabis component, and the cannabis component extract is directly used as a sample as it is. The component is measured and detected by an immunological measurement method using an antigen-antibody reaction. That is, according to the present invention, it is possible to limit the use of the alcohol component when extracting the cannabis component.

  As an organic solvent for cannabis component extraction, an alcohol having a small carbon number is used, preferably methanol, ethanol, or propanol. Among these, methanol is preferable. Moreover, you may use the solution for extraction which does not contain alcohol. That is, the alcohol in the cannabis component extraction solution is used in an amount of 0 to 40 wt%, preferably 0 to 30 wt%. The alcohol may be diluted with water, but is preferably diluted with a buffer or physiological saline. Further, it may be used preferably diluted with phosphate buffered saline (PBS-T) containing a surfactant or phosphate buffered saline (PBS) containing no surfactant. The aqueous alcohol solution for cannabis component extraction preferably contains a surfactant, but an aqueous alcohol solution that does not contain a surfactant can also be used. The surfactant is not limited, but Tween-20, Tween-80 and the like are preferably used.

  At this time, the higher the alcohol concentration, the better the extraction efficiency, but in order to carry out an antigen-antibody reaction, THC, THC-A or CBN is extracted at a concentration of several μg / mL or more, for example, 3 to 4 μg / mL or more. I can do it. Accordingly, it is possible to extract THC, THC-A, or CBN at a concentration that can be measured by antigen-antibody reaction even if extraction is performed with a solution containing a low concentration of alcohol or an extraction solution that does not contain alcohol. If the alcohol concentration when extracting the cannabis component is low, the cannabis component extract containing alcohol can be used as a specimen sample for immunological measurement using an antigen-antibody reaction directly.

  Extraction is carried out by placing cannabis plant pieces in an extraction solution containing alcohol and stirring, shaking, or standing. Stirring can be performed using a stirrer such as a vortex mixer. Moreover, you may extract by irradiating an ultrasonic wave. Stirring or vibration may be performed, for example, for 10 seconds to 60 minutes. Moreover, when extracting by standing, a cannabis plant piece is put into the solution for cannabis component extraction, and it should just stand for 10 seconds or more. Although the size of the cannabis plant piece is not limited, for example, it may be extracted as a 1 to 10 mm square piece. Moreover, you may extract after grind | pulverizing. The container used for extraction is not limited, and for example, a glass vial or a resin vial such as polypropylene may be used. Moreover, you may use the device which can extract a cannabis component and can also perform subsequent antigen antibody reaction.

The antibody is a specific antibody against the cannabis component, preferably an anti-THC antibody, an anti-THC-A antibody or an anti-CBN antibody. Since THC, THC-A and CBN have similar structures and immunologically cross-react, using either anti-THC antibody, anti-THC-A antibody or anti-CBN antibody, THC, THC -A and CBN can be measured. The antibody may be a complete antibody or a functional fragment of an antibody. Examples of the functional fragment include Fab antibody, F (ab ′) ₂ antibody, Fab / c antibody having one Fab and complete Fc, and the like. Further, it may be an antibody fragment or an artificial antibody that has a region to which Protein A binds and a region to which Protein G binds and has a structure capable of binding to an antigen. Examples thereof include antibody fragments and artificial antibodies having a VH region to which Protein A binds and a CH1 region to which Protein G binds. In the present invention, the term “antibody” includes functional fragments of such antibodies and artificial antibodies. The antibody may be a monoclonal antibody produced by an antibody-producing hybridoma or a fragment thereof, or an antibody prepared by genetic recombination technology based on the DNA information of the monoclonal antibody produced by the hybridoma. An example of such a hybridoma is hybridoma A-04, which is dated November 20, 2014, and is a NITE Patent Microorganisms Depositary (NITE). ) (No. 2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba, Japan, Room 122) is deposited internationally under the deposit number NITE BP-01970 (“Identification” is “A-04”). Among these antibodies, a Fab fragment (Fab antibody) having one heavy chain variable region and one heavy chain CH1 region and one light chain variable region and one light chain CL region is preferable. The animal from which the antibody is derived is not limited, and humans, mice, rats, rabbits, goats, horses, musk rats, sheep and the like can be used. The antibody may be a labeled antibody. Examples of labeled antibodies include antibodies labeled with fluorescent dyes, enzymes, chemiluminescent substances, and the like. Fluorescently labeled antibodies designed to change the presence or absence of fluorescence and change in fluorescence intensity when the antibody binds to the antigen and when the antibody does not bind to the antigen. Preferably used. That is, when the labeled antibody is not bound to the antigen, the fluorescent substance used for the labeling is quenched (quenched) so that it does not emit fluorescence or is in a state of generating fluorescence of a specific wavelength. When it is bound, the fluorescence generation state of the fluorescent dye can be changed. For example, a fluorescent dye that has been quenched when the antibody and the antigen are not bound to fluoresce when the antibody and the antigen are bound, or fluoresce when the antibody and the antigen are not bound. The fluorescent dye that has been used causes the wavelength of the fluorescence generated by the binding of the antibody and the antigen to shift. Examples of such an antibody include an antibody in which the fluorescence intensity is changed by a quenching dye (quencher), and an antibody in which the emission state of the fluorescent dye is changed by fluorescence resonance energy transfer (FRET). That is, in the present invention, an antibody that can change the fluorescence intensity when a cannabis component and an antibody against the cannabis component form a complex is used. (1) When antibody heavy chain variable region polypeptide and antibody light chain variable region polypeptide form a complex via the antigen, quenching is eliminated and fluorescence intensity increases. When the antibody and (2) the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide bind to the cannabis component as an antigen to form a complex, the complex of the antigen and the antigen binding protein A fluorescent dye quencher is formed, and when the complex of the antigen, the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide is formed, the fluorescent dye is more strongly quenched, thereby reducing the fluorescence intensity. An antibody is mentioned. When the former antibody is used, the concentration of the antigen to be measured and the fluorescence intensity of the fluorescent dye are positively correlated, and the concentration of the antigen to be measured and the fluorescence intensity of the fluorescent dye are positively correlated. It is called an antibody. When the latter antibody is used, the antigen concentration to be measured and the fluorescence intensity of the fluorescent dye have a negative correlation, and the antigen concentration to be measured and the fluorescence intensity of the fluorescent dye have a negative correlation. It is called an antibody.

  For example, an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide, wherein one or both of the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide are antibody heavy chain variable regions The antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide form a complex through the antigen by being labeled with a fluorescent dye that is quenched while being labeled to the polypeptide or antibody light chain variable region polypeptide Then, an antibody whose quenching is eliminated and the fluorescence intensity is increased can be suitably used. And an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide, wherein one or both of the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide are labeled with a fluorescent dye. When the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide are combined with a cannabis component that is an antigen to form a complex, the complex of the antigen and the antigen binding protein Use an antibody that becomes a quencher, and whose fluorescence intensity decreases when the complex of the antigen, the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide is formed, and the fluorescent dye is more strongly quenched be able to. In the present invention, such an antibody is referred to as Q-body (trademark), and Q-body (trademark) in which the concentration of the antigen to be measured and the fluorescence intensity of the fluorescent dye are positively correlated with the concentration of the antigen to be measured Q-body (trademark) in which the fluorescence intensity of the fluorescent dye has a negative correlation can be used. Q-body (trademark) is also referred to as a Fab-type complex or Fab-type antibody.

  In particular, a fluorescently labeled antibody that uses a tryptophan residue present in the VH region of the antibody as a quenching dye (quencher). There are tryptophan (W) residues at the 36th, 47th, and 103rd positions of the antibody VH region (according to the Kabat numbering system), and these tryptophan residues act as quenchers ( WO2011 / 061944). It is designed such that when bound to an antibody antigen labeled with a fluorescent dye, the fluorescent dye is located in the vicinity of the tryptophan residue and interacts with the tryptophan residue to quench the fluorescent dye. That is, when the fluorescently labeled antibody is not bound to the antigen, it is quenched and does not emit fluorescence. When an antibody with a positive correlation between the antigen concentration and the fluorescence intensity of the fluorescent dye is used, when the antigen binds to the antibody, the three-dimensional structure of the antibody changes, and the fluorescent dye located near tryptophan moves away from tryptophan. , No longer interacts with tryptophan, the quench is released and fluorescence is emitted. When the antigen is present, the antibody and the antigen are bound, the three-dimensional structure of the antibody is changed, the quenching of the fluorescent dye is released, and fluorescence is emitted. By measuring this fluorescence, the presence of the antigen can be detected, and the antigen can also be quantified by the fluorescence intensity. On the other hand, when an antibody in which the antigen concentration and the fluorescence intensity of the fluorescent dye have a negative correlation is used, the complex of the antigen and antibody acts as a quencher on the fluorescent dye, and the fluorescent dye is further quenched to generate a fluorescent dye. The fluorescence intensity of the fluorescence to be weakened. In this case, the fluorescent dye used for labeling the antibody light chain variable region polypeptide and / or antibody heavy chain variable region polypeptide of the antibody is located in the antigen-binding pocket of the antibody and is combined with tryptophan of the heavy chain variable region. Located in close proximity, interaction with tryptophan becomes stronger and quenched. When both the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide are labeled with a fluorescent dye, both fluorescent dyes enter the antigen-binding pocket of the antibody and there is interaction between the two fluorescent dyes. And quenching effect (H-dimer) between fluorescent dyes is obtained. In this case, the fluorescent dye used for labeling the antibody light chain variable region polypeptide is different from the fluorescent dye used for labeling the antibody heavy chain variable region polypeptide, and is an energy donor (donor) for fluorescence resonance energy transfer. When the antibody binds to the antigen, the orientation of both fluorescent dyes, ie, the energy donor and the energy acceptor, changes, and the energy of the donor dye becomes the energy acceptor (acceptor). Fluorescence resonance energy transfer (FRET) from the energy emitted by the donor to the energy acceptor does not occur, and the fluorescence intensity of the generated fluorescence is weakened. That is, it comprises an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide, and one or both of the antibody light chain variable region polypeptide and antibody heavy chain variable region polypeptide are labeled with a fluorescent dye. When measuring and detecting an antigen using an antibody, in addition to quenching with a tryptophan residue and quenching between fluorescent dyes, a quenching effect by a fluorescence resonance energy transfer (FRET) effect is obtained, resulting in a large quench. The fluorescent dye interacts with the antigen-antibody complex by hydrophobic interaction, electrostatic interaction, or the like, and the degree of quenching is increased.

  An antibody having a negative correlation between the antigen concentration and the fluorescence intensity of the fluorescent dye includes an antibody produced by hybridoma A-04 (Accession No. NITE BP-01970). The antibody light chain variable region polypeptide of the antibody and the anti-antibody A Q-body (trademark) in which the antigen concentration and the fluorescence intensity of the fluorescent dye have a negative correlation can be prepared by using the weight chain variable region polypeptide.

  The Q-body antibody heavy chain variable region polypeptide includes an amino acid sequence specific to the antibody heavy chain variable region encoded by exons of the V region, D region, and J region of the antibody heavy chain gene. Any amino acid sequence may be added to the N-terminal and / or C-terminal side of the amino acid sequence specific to the antibody heavy chain variable region. The amino acid sequence specific to the antibody heavy chain variable region is an amino acid sequence in which the 36th, 47th, or 103rd amino acid is tryptophan in the Kabat numbering system. preferable.

  The Q-body antibody light chain variable region polypeptide is not particularly limited as long as it contains an amino acid sequence specific to the antibody light chain variable region encoded by exons of the V region and J region of the antibody light chain gene. However, an arbitrary amino acid sequence may be further added to the N-terminal and / or C-terminal side of the amino acid sequence specific to the antibody light chain variable region. The amino acid sequence specific to the antibody light chain variable region is preferably an amino acid sequence in which the 35th amino acid is tryptophan in the Kabat numbering system.

  Antibody light chain variable region polypeptides, antibody light chain variable region polypeptides, and single chain antibody polypeptides comprising both antibody light chain variable regions and antibody light chain variable regions are known chemical synthesis methods, genetic recombination Although it can be prepared using a technique, a method for degrading antibody molecules with a proteolytic enzyme, etc., among them, it is preferable to prepare by a gene recombination technique that can be prepared in a large amount by a relatively easy operation.

  The fluorescent dye for labeling Q-body is not particularly limited as long as it is quenched (quenched) in a state labeled with antibody heavy chain variable region polypeptide or antibody light chain variable region polypeptide. Rhodamine, coumarin , Cy, EvoBlue, oxazine, Carbopyronin, naphthalene, biphenyl, anthracene, phenenthrene, pyrene, carbazole and the like, and a fluorescent dye and derivatives of the fluorescent dye can be exemplified. Specifically, CR110: carboxyrhodamine 110 : Rhodamine Green (trade name), TAMRA: carbocytetremethlrhodamine: TMR, ATTO655 (trade name), BODIPY FL (trade name): 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s -indancene-3-propionic acid, BODIPY 493/503 (trade name): 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a, 4a-diaza-s-indancene-8-propionicacid , BODIPY R6G (trade name): 4,4-difluoro-5- (4-phenyl-1,3-butadienyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 558 / 568 (trade name) 4,4-difluoro-5- (2-thienyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 564/570 (trade name): 4,4-difluoro-5- styryl-4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 576/589 (trade name): 4,4-difluoro-5- (2-pyrrolyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 581/591 (trade name): 4,4-difluoro-5- (4-phenyl-1,3-butadienyl) -4-bora-3a, 4a- diaza-s-indancene-3-propionic acid, Cy3 (trade name), Cy3B (trade name), Cy3.5 (trade name), Cy5 (trade name), Cy5.5 (trade name), EvoBlue10 (trade name) , EvoBlue30 (trade name), MR121, ATTO 390 (trade name), ATTO 425 (trade name), ATTO 465 (trade name), ATTO 488 (trade name), ATTO 495 (trade name), ATTO 520 (trade name) ATTO 532 (trade name), ATTO Rho 6G (trade name), ATTO 550 (trade name), ATTO 565 (trade name), ATTO Rho 3B (trade name), ATTO Rho 11 (trade name), ATTO Rho 12 (trade name), ATTO Thio12 (trade name), ATTO 610 (trade name), ATTO 611X (trade name), ATTO 620 (trade name) ATTO Rho14 (trade name), ATTO 633 (trade name), ATTO 647 (trade name), ATTO 647N (trade name), ATTO 655 (trade name), ATTO Oxa12 (trade name), ATTO 700 (trade name), ATTO 725 (trade name), ATTO 740 (trade name), Alexa Fluor 350 (trade name), Alexa Fluor 405 (trade name), Alexa Fluor 430 (trade name), Alexa Fluor 488 (trade name), Alexa Fluor 532 (trade mark) Name), Alexa Fluor 546 (trade name), Alexa Fluor 555 (trade name), Alexa Fluor 568 (trade name), Alexa Fluor 594 (trade name), Alexa Fluor 633 (trade name), Alexa Fluor 647 (trade name) , Alexa Fluor 680 (trade name), Alexa Fluor 700 (trade name), Alexa Fluor 750 (trade name), Alexa Fluor 790 (trade name), Rhodamine Red-X (trade name), Texas Red-X (trade name) , 5 (6) -TAMRA-X (trade name), 5TAMRA (trade name), SFX (trade name), among them CR110 and TAMRA, which are rhodamine fluorescent dyes, and oxazine fluorescent Particularly preferred is ATTO655, which is a dye.

  Q-body is described in Japanese Patent No. 5043237 and WO2013 / 065314.

  The immunological measurement method using the antigen-antibody reaction is not limited, and can be measured by a known ELISA, immunochromatography or the like. A cannabis component extracted from a cannabis plant piece and a specific antibody against the cannabis component may be mixed to detect an antigen-antibody complex. When the above Q-body is used, the fluorescence generated when the antibody and the cannabis component are combined may be measured. The antigen-antibody reaction system for immunological measurement may be a liquid system or a solid phase system. The solid phase system uses a dried antibody. As the dry antibody, a freeze-dried antibody or an antibody immobilized on a carrier such as a microtiter plate can be used. An antibody solution or a dry antibody may be placed in a container in which an antigen-antibody reaction is performed, and a cannabis component extract extracted from a cannabis plant piece may be added to the container.

  A cannabis component extract containing or not containing an alcohol component during the antigen-antibody reaction is used as a sample sample. However, if the reaction system contains a high concentration of alcohol when the antigen-antibody reaction occurs, the antigen-antibody reaction is inhibited. The The alcohol final concentration during the antigen-antibody reaction is 30 wt% or less, preferably 25 wt% or less, more preferably 20 wt% or less, more preferably 15 wt% or less, more preferably 10 wt% or less, more preferably 7.5 wt% or less, Preferably it is 5 wt% or less, and particularly preferably 0 wt%.

  In the method of the present invention, when a cannabis component is extracted from a cannabis plant piece using an extraction solution that may contain alcohol, and a solution containing alcohol is used as the extraction solution, the cannabis component extract contains alcohol. Therefore, the final alcohol concentration when the cannabis component extract and the antibody are added is 30 wt% or less, preferably 25 wt% or less, more preferably 20 wt% or less, more preferably 15 wt% or less. It is necessary to adjust the alcohol concentration of the cannabis component extraction solution so that it is more preferably 10 wt% or less, more preferably 7.5 wt% or less, more preferably 5 wt% or less, and particularly preferably 0 wt%. When using a dried antibody or a solid-phased antibody, the alcohol concentration in the cannabis component extraction solution is 30 wt% or less, preferably 25 wt% or less, more preferably 20 wt% or less, more preferably 15 wt% or less, More preferably, it may be 7.5 wt% or less. In addition, when an antigen-antibody reaction is performed by mixing the cannabis component extract and the antibody solution at a volume ratio of 1: 1, the alcohol concentration of the cannabis component extraction solution is 30 wt% or less, more preferably 20 wt% or less, more preferably Is 15 wt% or less, more preferably 10 wt% or less, particularly preferably 0 wt%, and when an antigen-antibody reaction is carried out by mixing the cannabis component extract and the antibody solution at a volume ratio of 1: 2, The alcohol concentration of the mixture is 30 wt% or less, preferably 22.5 wt% or less, more preferably 15 wt% or less, and even more preferably 0 wt%, and the cannabis component extract and the antibody solution are mixed at a volume ratio of 1: 3 for antigen-antibody reaction. In the case of carrying out, the alcohol concentration of the cannabis component extraction liquid may be 30 wt% or less, preferably 20 wt% or less, more preferably 0 wt%. Usually, at the time of antigen-antibody reaction, the cannabis component extract and the antibody solution are mixed at a volume ratio of 1: 1 to 1: 3. Therefore, the alcohol concentration in the cannabis component extraction liquid in consideration of both the cannabis component extraction efficiency and the antigen-antibody reaction is 30 wt% or less, preferably 22.5 wt% or less, more preferably 20 wt% or less, more preferably It is 15 wt% or less, more preferably 10 wt% or less, and particularly preferably 0 wt%.

  When performing immunological measurements, cannabis components can be extracted from cannabis plant pieces and mixed with antibodies against cannabis components to cause an antigen-antibody reaction. Test devices that extract cannabis components and cause an antigen-antibody reaction May be used. Examples of such a device include a device having a container part for extracting a cannabis component from a cannabis plant piece, and a container part for mixing a cannabis component and an antibody against the cannabis component to cause an antigen-antibody reaction. The device may be referred to as a cannabis component testing device.

  Hereinafter, a test device for extracting a cannabis component and performing an antigen-antibody reaction, a method for extracting a cannabis component using the device, and a method for performing an antigen-antibody reaction by mixing a cannabis component extract with an antibody against the cannabis component To do.

  The device includes a first container and a second container. The device may be an integrated device in which a first container and a second container are continuous through a partition wall, and a predetermined amount of liquid can be moved and added from the first container to the second container. The first container and the second container may be separate devices that are not continuous. The first container contains a solution having an alcohol concentration of 30 wt% or less, and the second container contains an antibody against the cannabis component for detecting an antigen by an antigen-antibody reaction using the cannabis component as an antigen. The antibody may be liquid or dried. Examples of the dried product include freeze-dried antibody, antibody immobilized on a second container, and the like. In the first container, the cannabis component is extracted from the cannabis plant piece, and the extract containing the cannabis component is added to the second container by moving a constant volume, and an antigen-antibody reaction occurs in the second container.

  The capacity of the first container is about 1.2 to 4.8 mL, the capacity of the second container is about 50 to 200 μL, and the overall size of the device is about 54 to 216 mm in length and about 7 to 26 mm in outer diameter. .

  The device includes a constant-volume transfer mechanism having a mechanism for extracting a cannabis component into a second container after extracting the cannabis component using an extraction solution containing alcohol in the first container. It may be. A target plant test piece suspected of being a cannabis plant piece is placed in a first container containing an extraction solution, and extracted by stirring, shaking or standing still.

  The first container and the second container of the device have a cylindrical shape, and the second container is mounted below the first container. A measuring member is fitted on the inner peripheral side of the first container, and the measuring member is movable in a direction in which the measuring member comes into contact with and separates from the first container. In addition, a cap member is inserted into the upper opening of the first container, and the cap member moves in a direction of contacting and separating from the measuring member by rotating 90 degrees and pushing in or removing 9 mm. Is possible. When the cannabis component is extracted with the cannabis component testing device, the cap member of the testing device is rotated and removed, the test sample is put, the cap is then closed, and the cannabis component is extracted.

  A liquid circulation opening through which liquid flows is formed at the bottom of the measuring member, and a measuring rod provided with a measuring groove for measuring the liquid projects from the bottom of the measuring member.

  After extracting the cannabis component in the first container, the cannabis component extract containing the cannabis component is accommodated in the measuring groove through the liquid flow opening of the measuring member and approaches the cap member By moving the cap member, the cap member comes into contact with the measuring member, and further, the measuring member is moved in the direction approaching the second container by moving the cap member in the direction approaching the measuring member. As a result, a predetermined amount of cannabis component extract contained in the measuring groove of the measuring member is added to the second container and mixed with the antibody against the cannabis component contained in the second container, and the antigen antibody A reaction occurs.

  That is, by using this device, the cannabis component extract extracted in the first container is accommodated in the measuring groove through the liquid flow opening of the measuring member. Thereafter, by moving the cap member in a direction approaching the measuring member, the cap member abuts on the measuring member, and further, moving the cap member in a direction approaching the measuring member, A predetermined amount of the cannabis component extract that moves in the direction approaching the second container and is accommodated in the measuring groove of the measuring member can be added to the antibody against the cannabis component contained in the second container.

  The metering member includes a metering rod, and the metering rod is configured to slide in a liquid-tight state along a metering side wall formed inside the first container.

  With this configuration, the measuring rod of the measuring member slides in a liquid-tight state along the measuring side wall formed on the inner periphery of the first container, so that it is once accommodated in the measuring groove. A predetermined amount of cannabis component extract can always be mixed with an antibody against the cannabis component contained in the second container without leakage of the predetermined amount of cannabis component extract.

  FIG. 2 shows a longitudinal sectional view of a testing device for extracting the above-mentioned cannabis component and causing an antigen-antibody reaction. The test device 10 includes a first container 12 that contains an extraction solution L1 for extracting a cannabis component, and a second container that contains an antibody L2 against the cannabis component below the first container. A container 14 is mounted.

  In addition, a measuring member 16 that can move in the direction of contacting and separating with respect to the second container 14 is fitted to the inner peripheral side of the first container 12.

  Further, a cap member 18 that is movable in the direction of contacting and separating with respect to the measuring member 16 is inserted into the upper opening 12 a of the first container 12. The first container 12 includes a substantially cylindrical container main body 20, and a measuring side wall 22 having a smaller diameter than the diameter of the container main body 20 is formed at the bottom of the container main body 20. Yes. Further, an engaging portion 24 whose diameter is increased again extends from the lower end of the measuring side wall 22, and an engaging protrusion 24 a is formed on the outer periphery of the engaging portion 24.

  In addition, a positioning projecting portion 26 for positioning the measuring member 16 when the measuring member 16 is fitted to the inner peripheral side of the first container 12 is formed on the inner wall 20a of the container body 20. ing.

  The inner wall 20a near the upper opening 12a of the first container 12 has a locking projection 28 for locking the cap member 18 when the cap member 18 is attached to the upper opening 12a. Is formed.

  A guide groove 30 that guides the cap member 18 when the cap member 18 is moved in a direction approaching the measuring member 16 is formed in the inner wall 20a in the vicinity of the upper opening 12a of the first container 12. Yes.

  As shown in FIG. 2, the second container 14 includes a second container body 14a having a substantially cylindrical shape, and the antibody L2 against the cannabis component is accommodated inside the second container body 14a. The bottomed cylindrical antibody accommodating part 34 is formed.

  Further, the upper portion of the main body 14a of the second container 14 is engaged with an engaging protrusion 24a formed on the outer periphery of the container main body portion 20 of the first container 12 when mounted below the first container 12. An engagement slit 14b is formed.

  Furthermore, two opposing slit openings 36 are formed in the lower portion of the main body 14a of the second container 14, and, for example, light is irradiated through these slit openings 36 to form the second It is comprised so that the test | inspection of the antigen antibody reaction of the container 14 can be performed.

  It should be noted that depending on the intended use of the cannabis component testing device 10 of the present invention, it is possible not to provide such a slit opening 36.

  As shown in FIG. 2, the measuring member 16 includes a substantially cylindrical measuring member main body 38, and a liquid circulation opening 40 through which liquid flows is formed at the bottom of the measuring member main body 38. ing.

  A measuring rod 44 is provided at the lower end of the measuring member main body 38 of the measuring member 16, and the measuring rod 44 is disposed along the measuring side wall 22 formed on the inner periphery of the first container 12. It is configured to slide in a dense state.

  The outer diameter of the measuring member main body 38 of the measuring member 16 is formed to be substantially the same as or slightly smaller than the inner diameter of the container main body portion 20 of the first container 12, and the measuring member 16 is the container of the first container 12. It slides along the inner wall 20a of the main body 20 and can move in the direction of contact with and away from the second container 14.

  On the other hand, as shown in FIG. 2, the cap member 18 is spaced apart from the substantially cylindrical cap member main body 52, the upper surface 54, and the cap member main body 52, and extends downward from the outer peripheral end of the upper surface 54. And an operating member 56 provided.

  In this case, the outer diameter of the cap member main body 52 is formed to be substantially the same as or slightly smaller than the inner diameter of the container main body portion 20 of the first container 12, and is in contact with the measuring member 16 as will be described later. It can move in the direction of separation.

  In addition, even when the cannabis component testing device 10 is accidentally tilted, the cap member main body 52 prevents the extraction liquid L1 accommodated in the container main body portion 20 of the first container 12 from leaking to the outside. It is desirable that the outer diameter of the first container 12 is dimensioned so as to be liquid-tightly sealed with respect to the inner diameter of the container body 20 of the first container 12.

  Further, the inner diameter of the operation member 56 is substantially the same as or slightly larger than the outer diameter of the container body 20 of the first container 12 so as to be able to move in the direction of contact with and separating from the measuring member 16. It is formed as follows.

  Further, the upper end 12b of the first container 12 abuts on the lower surface 54a of the upper surface 54 located between the cap member main body 52 and the operation member 56 and functions as a stopper, so that the measuring member 16 is further removed. The second container 14 is configured not to move in the approaching direction.

  Although not shown, the cap member main body 52 of the cap member 18 is formed with a guide projecting portion that fits and slides in the guide groove 30 formed on the inner wall of the first container 12. .

  The cannabis component testing device 10 of the present invention configured as described above is used as follows, for example.

  First, as shown in FIG. 2, the cannabis component testing device 10 of the present invention is in a pre-assembled state before use.

  That is, by engaging the engagement slit 14b on the upper part of the main body 14a of the second container 14 with the engagement protrusion 24a formed on the outer periphery of the engagement portion 24 of the first container 12, the second container 14 is attached to the engaging portion 24 below the first container 12. In addition, the antibody L2 is stored in advance in the antibody storage portion 34 of the second container 14.

  Further, as shown in FIG. 2, a measuring member 16 that can move in the direction of contact with and away from the second container 14 is fitted on the inner peripheral side of the container main body 20 of the first container 12. Has been.

  That is, when the measuring member 16 is fitted to the inner peripheral side of the first container 12, the lower end 38 a of the measuring member main body 38 of the measuring member 16 is positioned on the inner wall 20 a of the container main body 20. The measuring member 16 is positioned at the initial position by contacting the portion 26 and functioning as a stopper.

  In this state, the lower seal portion 48 of the measuring rod 44 of the measuring member 16 is in a state of being fitted to the measuring side wall 22 formed on the inner periphery of the first container 12 so as to be sealed in a liquid-tight manner. It has become.

  Thereby, the cannabis component extract L1 to be mixed accommodated in the main body part 20 of the first container 12 is the liquid flow opening 40 formed in the bottom part of the measuring member main body 38, and the measuring side wall of the container main body 20 22 is configured so as not to be mixed with the antibody L2 accommodated in the antibody accommodating part 34 of the second container 14 through the opening surrounded by the numeral 22.

  In this state, the measuring member 16 is fitted into the upper opening 12 a of the first container 12. And it forms in the latching protrusion part 28 formed in the inner wall 20a of the vicinity of the upper opening part 12a of the 1st container 12 so that it may protrude outside in the approximate middle of the cap member main body 52 of the cap member 18. The locking projection 52a comes into contact with each other and functions as a stopper, and the cap member 18 is locked at the initial position.

  Next, the cap member 18 is fitted into the upper opening 12 a of the first container 12 again.

  Each time the cap member 18 is rotated, the guide groove 30 formed on the inner wall 20a near the upper opening 12a of the first container 12 and the guide protrusion formed on the cap member main body 52 of the cap member 18 are provided. Match the parts.

  Then, the cap member 18 is held on the second container 14 by grasping the operation member 56 of the cap member main body 52 with a finger or the like and pressing it downward, or by pressing the upper surface 54 of the cap member main body 52 downward. Move in the direction.

  As a result, the lower end 52b of the cap member main body 52 of the cap member 18 abuts on the upper end of the measuring member main body 38 of the measuring member 16, and the lower end 38a of the measuring member main body 38 of the measuring member 16 and the inner wall of the container main body portion 20. The contact state with the positioning protrusion 26 formed on 20a is released, and the measuring member 16 is moved in the direction of the second container 14, that is, downward.

  In this state, the cannabis component extract L1 to be mixed accommodated in the container body 20 of the first container 12 passes through the liquid circulation opening 40 formed at the bottom of the measuring member body 38 of the measuring member 16. The metering member 16 enters the metering groove 50 formed between the upper seal portion 46 and the lower seal portion 48 of the metering rod 44 of the metering member 16.

  Then, by further moving the cap member 18 in the direction of the second container 14, the upper sealing portion 46 of the measuring rod 44 of the measuring member 16 is formed on the inner side wall 22 of the first container 12. And is sealed in a liquid-tight manner.

  Accordingly, the measuring groove 50 formed between the upper seal portion 46 and the lower seal portion 48 of the measuring rod 44 of the measuring member 16, and the measuring side wall 22 formed on the inner periphery of the first container 12. A space is formed between the two.

  Thereby, the cannabis component extract L1 accommodated in the space is weighed.

  Subsequently, by moving the cap member 18 further in the direction of the second container 14, the measuring member 16 is moved further downward in the direction of the second container 14.

  As a result, the lower seal portion 48 of the measuring rod 44 of the measuring member 16 is separated from the lower end of the measuring side wall 22 formed on the inner periphery of the first container 12, and an opening is formed. .

  As a result, the cannabis component extract L1 accommodated and weighed in the space enters the antibody accommodating part 34 of the second container 14 through this opening, and the antibody accommodating part 34 of the second container 14 Mixing with the antibody L2 contained in is started.

  Then, by further moving the cap member 18 in the direction of the second container 14, the measuring member 16 is further moved downward in the direction of the second container 14 to the final position (mixing completion position).

  Thereby, the upper seal portion 46 of the measuring rod 44 of the measuring member 16 acts as a piston. Thereby, the measuring groove portion 50 formed between the upper seal portion 46 and the lower seal portion 48 of the measuring rod 44 of the measuring member 16, that is, the cannabis component extract L1 accommodated and measured in the space, It enters the antibody container 34 of the second container 14 and is mixed with the antibody L2 stored in the antibody container 34 of the second container 14.

  In the state of this final position, the upper end 12b of the first container 12 abuts on the lower surface 54a of the upper surface 54 located between the cap member main body 52 and the operation member 56, and functions as a stopper. No more 16 moves relative to the second container 14 in the approaching direction.

  Further, in this final position, the upper seal portion 46 of the measuring rod 44 of the measuring member 16 is fitted to the measuring side wall 22 formed on the inner periphery of the first container 12, and is liquid-tight. Is sealed.

  Accordingly, the cannabis component extract L1 accommodated in the container main body 20 of the first container 12 enters the antibody accommodating part 34 of the second container 14 further, and the antibody accommodating part of the second container 14 34 is configured not to be mixed with the antibody L2 accommodated in 34.

  According to the testing device 10 of the present invention configured as described above, the measuring groove portion 50 of the measuring member 16, that is, the predetermined amount of liquid stored in the space is transferred to the antibody storing portion of the second container 14. It can be mixed with the antibody L2 accommodated in 34.

  In the inspection device 10 of the present invention, the materials of the first container 12, the second container 14, the measuring member 16, and the cap member 18 are not particularly limited, and examples thereof include metals such as glass and aluminum. It can be made of a resin or the like. In this case, it is desirable to use a resin from the standpoint of metering and operability, and the resin that can be used is not particularly limited. For example, polyethylene resin (PE), polypropylene resin (PP), polyethylene terephthalate resin (PET) Etc. can be used.

  In addition, when optically testing or measuring the mixture, it is useful to use polystyrene resin (PS) or acrylic resin (PMMA) for the second container 14.

  The first container 12, the second container 14, the measuring member 16, and the cap member 18 can be opaque, translucent, and transparent.

  In the second container, the cannabis component extract and the antibody against the cannabis component are mixed to cause an antigen-antibody reaction.

  A target plant test piece suspected of being cannabis is placed in a first container containing a cannabis component extracting solution containing alcohol of the cannabis component testing device, and the cannabis component is extracted. The extract containing the cannabis component in the first container is transferred and added to the second container containing the antibody against the cannabis component using the mechanism of the device to cause the antigen-antibody reaction. The antibody in the second container may be contained as a dry product such as a lyophilized product or may be contained as an antibody solution. When included as an antibody solution, the mixing ratio (volume ratio) of the cannabis component extract containing alcohol and the antibody solution is 1: 1 to 1: 5, preferably 1: 2 to 1: 4, more preferably 1: 3. is there. For example, 1 to 5 mL, preferably 2 to 3 mL, and more preferably 2.4 mL of cannabis component extract is stored in a first container, and plant pieces suspected of containing cannabis are placed and extracted, and then the extract is added. Transfer 10 to 100 μL, preferably 20 to 50 μL, more preferably 25 μL to the second container. At this time, the cannabis component may be contained in the extract at a concentration of 4 μg / mL or more, preferably 4 to 400 μg / mL. At this time, 25 to 100 μL, preferably 50 to 100 μL, more preferably 75 μL of an antibody solution containing an antibody against the cannabis component may be placed in the second container (FIG. 7). As a result, when the cannabis component concentration in the cannabis component extract in the first container is 4 to 400 μg / mL, the cannabis component concentration when the antigen-antibody reaction is performed in the second container is 1 to 100 μg / mL. Become.

  The antigen-antibody reaction is carried out at 4-44 ° C., preferably 25-35 ° C., immediately after the reaction for 3 minutes, preferably 3 minutes or more.

  After the antigen-antibody reaction, the antigen-antibody complex formed in the reaction solution is detected. For example, when a fluorescently labeled antibody is used as the antibody, the fluorescence generated from the complex of the antigen and antibody may be measured. When Q-body is used as the antibody, the measurement can be performed by measuring the reaction solution after the antigen-antibody reaction in the second container with a fluorescence tester.

  As the fluorescence inspection device, any device can be used as long as it can detect fluorescence of a specific wavelength. For example, the portable fluorescence tester shown in FIG. 3 can be used. After extracting a cannabis component from a cannabis plant piece using the above cannabis component testing device and causing an antigen-antibody reaction, fluorescence may be measured with a fluorescence tester using the reaction solution as a sample. At this time, the second container portion containing the antigen-antibody reaction solution of the cannabis component test device can be inserted into the tester for measurement.

  The inspection device includes at least a light source that emits excitation light that can excite a fluorescent substance in a liquid phase object in the container to emit fluorescence, and a detector that detects the fluorescence emitted by the fluorescent substance. And a portable fluorometer equipped with an optical system that guides excitation light from the light source to the liquid phase object in the container and guides fluorescence from the liquid phase object to the detector. The inspection device includes a housing that integrally holds a container, a light source, a detector, and an optical system. FIG. 6 shows a measurement system of the fluorescence tester.

  FIG. 3 shows a schematic perspective view of the portable fluorescence tester, and FIG. 4 shows the internal configuration. As shown in FIG. 3, the fluorescence tester has a flat, substantially rectangular parallelepiped box shape as a whole. Since it is portable, its size is about the size of a person's palm or slightly larger. On the front surface of the housing 62 of the fluorescence tester, a display unit 63 for displaying the operation state of the photometer and measurement results, and several operation buttons 64 to 69 are provided.

  A part of the upper surface of the housing 62 of the fluorescence tester is an open / close lid 61. When the opening / closing lid 61 is opened, a reagent cell mounting portion 60 is formed in the housing 62. The reagent cell mounting portion 60 is a frame-shaped portion that matches the size and shape of the reagent cell 70.

  The reagent cell 70 is formed of a material that sufficiently transmits excitation light and fluorescence. Specifically, a material made of glass such as borosilicate glass, quartz, or sapphire, or a resin such as PMMA (acrylic resin), polystyrene, or COC (cyclic olefin copolymer) is used as the material of the reagent cell 70. . What is necessary is just to add the reaction liquid after making antigen-antibody reaction to the reagent cell 70, and to measure fluorescence.

  When the reagent cell 70 is attached to the fluorescence tester, as shown in FIG. 3, the open / close lid 61 is opened and the sample cell 70 is inserted into the insertion hole of the reagent cell attachment portion 60. The reagent cell 70 is mounted on the reagent cell mounting portion and is held at a predetermined position. Thereafter, the opening / closing lid 61 is closed.

  The light source 72 emits light (excitation light) that can excite the fluorescent substance in the liquid phase object to emit fluorescence. As the light source, laser light, LED light, or the like can be used. Preferably, an LED lamp is used. For example, an LED that emits green light having a wavelength of 525 nm may be used. A thing can be used suitably.

  The optical system of the fluorescence tester guides excitation light from the light source 72 to the cell portion of the reagent cell 70 and guides fluorescence from the liquid phase object of the cell to the detector 76. The detector 76 is appropriately selected from those using a photodiode or a phototube, and a photodiode is preferably used. In the case of the inspector having the internal structure shown in FIG. 4, the optical system includes a lens 71 that collects light from the light source 72, a dichroic mirror 75 for bending the optical path and selecting light, and a filter disposed on the optical path. (Excitation filter 73 and fluorescent filter 74) and the like. A detector 76 is disposed at a position opposite to the reagent cell mounting portion 60 with the dichroic mirror 75 interposed therebetween. The fluorescence tester further includes a battery 77 for supplying power and an arithmetic unit 78. The computing device can calculate the cannabis component concentration in the sample from the detected fluorescence intensity and display it on the display unit 63. The arithmetic device may be provided with a storage device, and an equation in which the fluorescence intensity and the cannabis component concentration are stored in advance in the storage device, and the cannabis component concentration is calculated based on the equation. The general expression is, for example, an expression representing a calibration curve created in advance.

  By using such a small tester, it becomes possible to detect the cannabis component on-site at the acquisition site without carrying the acquired sample to the laboratory for measurement.

  The present invention further includes a cannabis component test kit including the above-described test device, or a cannabis component test kit including the above-described test device and a fluorescence tester.

  The cannabis component is detected as follows using the test device for extracting the cannabis component and performing an antigen-antibody reaction and the fluorescence tester.

  Place the target plant test piece suspected of being cannabis into the first container of the cannabis component inspection device containing the cannabis component extraction solution containing alcohol at a concentration of 30 wt% or less, and stir, vibrate, or stand still Extract cannabis ingredients. By the mechanism of the cannabis component testing device, the cannabis component extract in the first container is moved and added to the second container of the cannabis component testing device in which antibodies against the cannabis component are accommodated. Thereafter, the antigen-antibody reaction proceeds in the second container.

  After the antigen-antibody reaction is performed, the reaction solution is transferred to a reagent cell of a fluorescence tester and measurement is performed. At this time, the second container of the cannabis component testing device can also be used as a reagent cell.

  When Q-body is used as an antibody, when cannabis plant pieces are contained in the plant pieces, the antibody binds to the cannabis component, which is an antigen, so that fluorescence is emitted and the presence of cannabis plant pieces is detected. can do.

  According to the method of the present invention, it is possible to inspect cannabis, which is an illegal drug, on site using a simple fluorescence tester at a site such as customs.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example 1 Examination of extraction efficiency from cannabis leaves using THC and THC-A alcohol (1) Examination of methanol concentration during extraction In the antigen-antibody reaction, the reaction can be confirmed if cannabis components of 4 μg / mL or more can be extracted. it can. The concentration of methanol used for extraction was changed, and it was confirmed whether cannabis components of 4 μg / mL or more could be extracted.

  As a solution for extracting cannabis components from cannabis leaves, PBS-T solution (phosphate buffered saline + interface) containing 10 wt%, 30 wt% or 40 wt% methanol or no methanol (0 wt%) Activator Tween 20 0.05% + preservative sodium azide 0.05%) was used. Sodium azide in PBS-T is a preservative for long-term storage and does not participate in antigen-antibody reaction.

  The solution was placed in a polypropylene resin container. Twenty cannabis leaves were prepared, the leaves were made into 5 mm square sections, about 20 mg in weight were put into the container and immersed in the solution, and the container was covered and vibrated up and down. Immersion is performed for 3 minutes, and 50 μL of the extract is quantified by HPLC (high performance liquid chromatography). The immersion time of 3 minutes was set as an allowable time for on-site inspection.

  FIG. 8 shows an outline of a method for extracting cannabis components.

HPLC (high performance liquid chromatography) measurement conditions were as follows.
(a) Column
XBridge BEH C18 Intelligent Speed (IS) Column, 130Å, 2.5 μm, 4.6 mm X 20 mm, 1 / pkg (Waters Part number: 186003088)
(b) Sample Injection: 50μl (stock solution)
(c) Buffer A: 0.1% TFA (trifluoroacetic acid)
Buffer D: Acetonitrile
(d) Elution (conditions for running buffer solution for elution)
The buffer A is flowed from the beginning, and the ratio of the buffer D is gradually changed.
0.00 [min] −5.00 [min], D conc 0% -100% gradient
5.00 [min] −6.00 [min], D conc 100% -0% gradient
6.01 [min] -11.00 [min], D conc 0%
11.00min, Stop
(e) Flow-rate: 1.5 mL / min, Temp: 30 ° C
Detection was based on the absorption peak at 228 nm. A high peak height indicates a large amount of detection.

  Five samples were prepared at each methanol concentration, and the concentration was quantified (N = 5).

  As a result, the average extraction amount of cannabis components was 8.7μg / mL, 9.3μg / mL, and 7.6μg / mL in the PSB-T solutions with methanol concentrations of 0wt%, 10wt%, and 30wt%, respectively. Also exceeded the minimum extraction of 4 μg / mL. It was also found that cannabis components can be extracted even at a methanol concentration of 0 wt%, and that there is no difference compared to when methanol concentrations are 10 wt% and 30 wt%.

  From the results of Example 2 described later, it was found that when the methanol concentration was 40 wt%, the antibody was denatured and hindered measurement. When the methanol concentration is 0 wt%, the antibody activity is maintained higher than when the methanol concentrations are 10 wt% and 30 wt%, and the cannabis component can be measured without denaturing the antibody.

  The smaller the amount of methanol, the greater the effect that the progress of the antigen-antibody reaction is maintained high without the antibody being denatured.

(2) Examination of Cannabis Component Extraction with Pure Water and PBS-T Solution Cannabis component was extracted from cannabis leaves using pure water and PBS-T solution in the same manner as (1), and the concentration was determined. (n = 3).

  As a result, the average extraction amount of the cannabis component was 3.4 μg / mL with pure water and 7.4 μg / mL with PBS-T solution, and the extraction concentration was more than twice that of pure water with PBS-T solution. For the extraction of cannabis components, a PBS-T solution containing no methanol is preferable rather than pure water containing no methanol. However, when pure water is used as the extract, the amount of cannabis used for the antigen-antibody reaction can be extracted although the extraction amount is inferior to that of the PBS-T solution.

  As a result of Example 1, it was found that cannabis leaf was introduced as a test piece into a solution having an alcohol component of 30 wt% or less, and the components of the test piece could be extracted into the solution.

Example 2 Effect of methanol on measurement of cannabis components using antigen-antibody reaction Q-body (T-3 antibody), an antibody obtained by labeling antibodies against THC and THC-A, which are cannabis components, with a fluorescent dye It was. Antibody Q-body against THC, which is a cannabis component, was prepared by the method described in WO2013 / 065314. That is, by the method described in Example 1 of WO2013 / 065314, a light chain variable region (VL; SEQ ID NO: 1) labeled with a TAMRA of an antibody against cannabis component THC and an antibody light chain constant region (Cκ; SEQ ID NO: 2) And double-label Fab type of the same color comprising a polypeptide comprising a heavy chain variable region (VH; SEQ ID NO: 3) and a heavy chain constant region (CH1; SEQ ID NO: 4) for cannabis component THC labeled with TAMRA A complex was synthesized and used as the Q-body. Q-body was used at a concentration of 16 nM in PBS containing 0.5% Tween20 and 2% BSA.

  A PBS-T solution containing THC at a concentration of 100 μg / mL and methanol at a concentration of 60 wt%, 50 wt%, 40 wt%, 30 wt%, 20 wt% or 10 wt% was used as the antigen solution.

  30 μL of the antibody solution and 30 μL of the antigen solution were mixed to cause an antigen-antibody reaction. The final methanol concentration during the antigen-antibody reaction was 30 wt%, 25 wt%, 20 wt%, 15 wt%, 10 wt% or 5 wt%, and the final concentration of Q-body was 8 nM. Antigen-antibody reaction was performed at 30 ° C. Each was measured at n = 3.

  The fluorescence of the reaction solution was measured using a fluorescence plate reader (Moleculardevices, SpectraMax Paradigm S / N: 32 770 02-2092). The excitation wavelength and fluorescence wavelength during measurement were 530 nm and 580 nm, respectively.

  The results are shown in FIG. The horizontal axis of the graph of FIG. 9 indicates the relative fluorescence intensity, 1.00 or higher indicates that the antigen can be detected, and BR> 5, and less than 1.00 indicates that it cannot be detected. As shown in FIG. 9, when the final methanol concentration during the antigen-antibody reaction was 15 WT% or less, the antigen-antibody reaction occurred and THC could be measured.

  Furthermore, the measurement was performed by the same method as described above, using THC or THC-A as the antigen, and using an antigen solution (methanol final concentration 0 wt%) to which methanol was not added. Measurements were taken at n = 5.

  FIG. 10 shows the results of THC measurement, and FIG. 11 shows the results of THC-A measurement. Both THC and THC-A could be measured at a final methanol concentration of 25 wt% to 30 wt%.

Example 3 Measurement of Cannabis Component Using Antigen-Antibody Reaction Manufacture of anti-tetrahydrocannabinol (THC) hybridoma (1) Manufacture of hybridoma A mouse strain (BALB / c) is immunized with BSA-conjugated THC antigen (Genway Biotech) together with adjuvant, and the spleen is removed after increasing the serum titer. Then, cell fusion by myeloma cell NS-1 strain (P3.NS-1 / 1.Ag4.1) by PEG method (40%) was performed. Further, selection by HAT medium and selection by ELISA (details are shown in 2) yielded an anti-THC antibody (IgG1, kappa) -producing hybridoma. The resulting hybridoma A-04, dated November 20, 2014, NITE Patent Microorganisms Depositary (NITE Patent Microorganisms Depositary) (Kazusa Kamashichi, Kisarazu, Chiba, Japan) 5-8 Room 122) was deposited internationally under the deposit number NITE BP-01970 (“Identification” is “A-04”).

(2) Assay of monoclonal antibody produced by hybridoma Anti-THC antibody reactivity was measured by competitive ELISA (Enzyme-Linked ImmunoSorbent Assay).
(i) A 0.10 M carbonate buffer solution (pH 8.6) (100 ng / mL) of THC-BSA was dispensed (100 μL / well) into a 96-well ELISA plate and left overnight at room temperature.
(ii) The solution was removed by suction, and the plate was washed 3 times with PBS. PBS containing 2% skim milk was dispensed (300 μL / well) and allowed to stand at 37 ° C. for 1 hour.
(iii) The solution was removed by aspiration, and the plate was washed 3 times with PBS containing 0.05% (v / v) Tween 20 to obtain an antigen-immobilized plate.
(iv) On the plate of (iii), a 50% (v / v) ethanol solution of tetrahydrocannabinol (THC) or a similar compound (tetrahydrocannabinolic acid (THC-A) or cannabinol (CBN)) (25 μL / Then, the culture supernatant (100 μL / well) of hybridoma A-04 appropriately diluted with PBS containing 0.10% gelatin was mixed and incubated at 37 ° C. for 1 hour.
(v) Aspirate the solution and wash the plate 3 times with T-PBS, add peroxidase-labeled goat anti-mouse IgG (Fc specific) antibody (1.6 μg / 100 μL / well) and incubate at 37 ° C. for 30 minutes did.
(vi) The plate is again washed three times with PBS containing 0.05% (v / v) Tween 20 and contains the substrate solution {o-PD (0.04%) and H ₂ O ₂ [0.06% (v / v)] Citrate / phosphate buffer (pH 5.0)} (100 μL / well) was added and incubated at room temperature for 30 minutes.
(vii) Enzyme reaction stop solution (1.0 MH ₂ SO ₄ ) (100 μL / well) was added and mixed, and then the absorbance at 490 nm was measured with a microplate reader (Bio-Rad).

  The results are shown in FIG. As shown in FIG. 12, the obtained antibody showed affinity for THCA, THC and CBN.

2 Measurement using Fab type antibody (1) Fab type antibody (construction of expression vector)
The light chain variable region and the antibody light chain constant region (Cκ; SEQ ID NO: SEQ ID NO: 1) of the antibody against tetrahydrocannabinol (THC) prepared in 1 (Accession No. NITE BP-01970 (“Identification” is “A-04”)) 5), a DNA sequence encoding a polypeptide comprising 5) is given a DNA sequence of ProX tag (the base sequence corresponding to the 9th amino acid is TTT at the N-terminus, and MSKQIEVNFSNET; SEQ ID NO: 6) when translated; Further, a gene having a linker (SEQ ID NO: 7) and a FLAG tag DNA sequence at the C-terminus was incorporated into a pIVEX2.3d vector (Roche Diagnostics). It also encodes a polypeptide containing the heavy chain variable region (accession number NITE BP-01970 (“Identification” is “A-04”)) of the antibody against THC and the antibody heavy chain constant region (CH1; SEQ ID NO: 8). The DNA sequence of the ProX tag (the base sequence corresponding to the 9th amino acid is TAG, and when translated, MSKQIEVNXSNET (X is a fluorescently labeled amino acid); SEQ ID NO: 9) Furthermore, a gene having a linker (SEQ ID NO: 7) and a His tag DNA sequence added to the C-terminus was incorporated into a pIVEX2.3d vector (Roche Diagnostics). In these constructed expression vectors, a ProX tag (VH is labeled when translated and VL is unlabeled) is added to the N-terminus of the inserted VL or VH, and a His tag or FLAG tag is added to the C-terminus. It is designed as follows.

(Synthesis of Fab type antibody)
The reaction solution (60 μL) consists of 3 μL Enzyme Mix, 0.6 μL Methionine, 30 μL 2 × Reaction Mix, 20 μL E-coli Lysate, 2 μL of two types of plasmid DNA (200 ng each), 3 μL of fluorescently labeled aminoacyl-tRNAamber (480 pmol), 1.4 μL of Nuclease Free Water was added. CloverDirect (trade name) tRNA Reagents for Site-Directed Protein Functionalization (manufactured by Protein Express) was used as a fluorescently labeled aminoacyl-tRNA (TAMRA-X-AF-tRNAamber) for producing a fluorescently labeled protein. The reaction solution was allowed to stand at 20 ° C. for 2 hours for reaction to synthesize the protein, and then complex formation was completed by further reaction at 4 ° C. for 16 hours. After completion of the reaction, SDS-PAGE (15%) was performed using 0.5 μL of the reaction solution, and protein expression was observed with a fluorescence image analyzer (FMBIO-III; manufactured by Hitachi Software Engineering). Furthermore, Western blotting was performed using an anti-His tag antibody or an anti-FLAG tag antibody, and it was confirmed that the target fluorescently labeled antibody variable region-containing peptide was synthesized.

(Purification of fluorescently labeled Fab antibody)
The synthesized fluorescently labeled Fab type antibody was purified by anti-FLAG M2 affinity gel (Sigma Aldrich) or His-Spin Trap Column (GE Healthcare). The above reaction solution (60 μL) was applied to a column containing anti-FLAG M2 affinity gel, incubated for 15 minutes at room temperature, and then washed with a wash buffer (20 mM Phosphate buffer (pH 7.4) /0.5 M NaCl / 0.1% Polyoxyethylene (23) Lauryl Ether) was washed 3 times. Next, elution was performed 3 times with 200 μL of Elute buffer (20 mM Phosphate buffer (pH 7.4) /0.5 M NaCl / 100 μg FLAG peptide / 0.1% Polyoxyethylene (23) Lauryl Ether). Next, the eluate was applied to a His-Spin Trap Column. After 15 minutes of incubation at room temperature, washing was performed 3 times with Wash buffer (20 mM Phosphate buffer (pH 7.4) /0.5 M NaCl / 60 mM imidazole / 0.1% Polyoxyethylene (23) Lauryl Ether). Next, elution was performed 3 times with 200 μL of Elute buffer (20 mM Phosphate buffer (pH 7.4) /0.5 M NaCl / 0.5 M imidazole / 0.1% Polyoxyethylene (23) Lauryl Ether). Further, the eluate was Amicon Ultra-0.5 centrifugal filter 10 kDa (Millipore), buffer exchanged with PBS (+ 0.05% Tween20), and concentrated. The concentration of the purified sample was measured using a fluorescence image analyzer (FMBIO-III; manufactured by Hitachi Software Engineering).

  The resulting fluorescently labeled Fab antibody was as follows. The first four alphabets are abbreviations for each.

(v) HTLT type
Antibody consisting of antibody heavy chain variable region polypeptide labeled with TAMRA and antibody light chain variable region polypeptide labeled with TAMRA (same color double label)
In the cannabis component extraction method described in Example 1, the cannabis component was extracted using an extraction solution having a methanol concentration of 0 wt%, and the cannabis component was measured using the Fab antibody. The measurement was performed using the testing device shown in FIG. 2 and the fluorescence tester shown in FIG.

  The first container of the testing device was charged with 2.4 mL of a dilution buffer having an alcohol concentration of 0 as an extract. As a dilution buffer, PBS-T solution (phosphate buffered saline + surfactant Tween 20 0.05% + preservative sodium azide 0.05%) was used. Thereafter, the cannabis leaf was dried and finely crushed, and about 30 mg was weighed with a weighing jig and put into a first container to extract cannabis components.

  In the second container, the Fab antibody (10.7 nM, 75 μL) was put, and 25 μl of the cannabis component extracted in the first container was mixed in the second container. The reaction was performed at 25 ° C.

  The experiment was performed three times. Samples A, B and C were used in each experiment.

The voltage value, which is an indicator of the amount of fluorescence received by the light receiver of the fluorescence tester, was examined to confirm a decrease in fluorescence. Changes in the voltage values of samples A, B and C (fluorescence change) are shown.
(i) Immediately after mixing the fluorescence change of sample A 1404.7mV
After mixing 40 seconds (after reaction) 1369.93mV
(ii) Change in fluorescence of sample B
Immediately after mixing 1360.64mV
20 seconds after mixing (after reaction) 1336.65 mV
(iii) Immediately after mixing the fluorescence change of sample C 1279.11mV
40 seconds after mixing (after reaction) 1255.94 mV
As described above, the amount of fluorescence decreased before and after the antigen-antibody reaction.

  This result indicates that there is a negative correlation between antigen concentration and fluorescence intensity.

DESCRIPTION OF SYMBOLS 10 Test | inspection device 12 which extracts cannabis component and performs antigen-antibody reaction 1st container 12a Upper opening part 12b Upper end 14 Second container 14a Second container main body 14b Engagement slit 16 Measuring member 18 Cap member 20 1st Container main body portion 20a inner wall 20b outer peripheral side wall 22 measuring side wall 24 engaging portion 24a engaging protrusion 26 positioning protruding portion 28 locking protruding portion 30 guide groove 32 indicator member 34 antibody accommodating portion 36 slit opening portion 38 measuring member Main body 38a Lower end 40 Liquid flow opening 44 Measuring rod 46 Upper seal 48 Lower seal 50 Measuring groove 52 Cap member main body 52a Locking projection 52b Lower end 54 Upper surface 54a Lower surface 56 Operation member 60 Reagent cell mounting portion 61 Opening / closing lid 62 Housing 63 Display 64 to 69 Operation Button 70 Reagent Cell 71 Lens 72 Light Source 73 Excitation Filter 74 Fluorescent Fill 75 dichroic mirror 76 detector 77 battery 78 arithmetic unit

  INDUSTRIAL APPLICABILITY According to the present invention, an on-site cannabis component inspection method that can easily detect cannabis components in a short time can be provided. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (21)

  A method for extracting cannabis components, comprising placing a target plant test piece suspected of being cannabis in a solution having an alcohol concentration of 30 wt% or less and extracting the components of the test piece into the solution.   The method for extracting cannabis components according to claim 1, wherein a solution containing no alcohol is used.   The solution is a phosphate buffered saline (PBS-T) containing a surfactant, a phosphate buffered saline (PBS) containing no surfactant, or a pure water solution. Extraction method of cannabis ingredients.   The method for extracting cannabis components according to any one of claims 1 to 3, wherein the alcohol is selected from the group consisting of methanol, ethanol and propanol.   The cannabis component extraction method according to any one of claims 1 to 4, wherein the cannabis component is tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THC-A), or cannabinol (CBN).   The method for extracting a cannabis component according to any one of claims 1 to 5, wherein the target plant test piece includes any part selected from the group consisting of leaves, stems, roots, seeds, and petals. An inspection device used to detect cannabis components,
A first container and a second container;
The first container contains a solution having an alcohol concentration of 30 wt% or less,
The second container contains an antibody against the cannabis component,
Put the target plant test piece suspected of being cannabis in the first container, extract the components of the test piece into a solution having an alcohol concentration of 30 wt% or less,
A test device for detecting a cannabis component that causes an antigen-antibody reaction in a second container by adding a predetermined amount of the extract in the first container to the second container.   The test device for detecting a cannabis component according to claim 7, wherein a final alcohol concentration at the time of an antigen-antibody reaction occurring in the second container is 7.5 wt% or less.   The solution is a phosphate buffered saline (PBS-T) containing a surfactant, a phosphate buffered saline (PBS) containing no surfactant, or a pure water solution. Device for detecting cannabis components in food.   The test device for detecting a cannabis component according to any one of claims 7 to 9, wherein the alcohol is selected from the group consisting of methanol, ethanol and propanol.   The test for detecting the cannabis component according to any one of claims 7 to 10, wherein the cannabis component is tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THC-A), or cannabinol (CBN). Device.   The test for detecting a cannabis component according to any one of claims 7 to 11, wherein the target plant test piece includes any part selected from the group consisting of leaves, stems, roots, seeds, and petals. Device.   The antibody against the cannabis component comprises an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide, and one or both of the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide are anti-antibodies. The antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide are mediated through an antigen by being labeled with a fluorescent dye that is quenched in a state labeled with the body weight chain variable region polypeptide or the antibody light chain variable region polypeptide. The test device for detecting a cannabis component according to any one of claims 7 to 12, which is an antibody whose quenching is eliminated and fluorescence intensity is increased when a complex is formed. The antibody against the cannabis component comprises an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide, and one or both of the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide are fluorescent dyes When the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide are combined with a cannabis component that is an antigen to form a complex, the complex of the antigen and the antigen binding protein is A quencher of the fluorescent dye, wherein the antigen concentration and the fluorescence intensity of the fluorescent dye are negatively correlated, and a complex of the antigen, the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide is The cannabis according to any one of claims 7 to 12, which is an antibody whose fluorescence intensity decreases when the fluorescent dye is more strongly quenched when formed. Test devices for detecting.
  The antibody against the cannabis component is an antibody produced by a hybridoma that produces an antibody that binds to tetrahydrocannabinol (THC) or a derivative thereof deposited internationally under the deposit number NITE BP-01970. A test device for detecting the cannabis component according to claim 1.   Put a target plant test piece suspected of being cannabis in a solution with an alcohol concentration of 30 wt% or less, elute the components of the test piece into the solution, add the extract to the antibody against the cannabis component, and antigen antibody A method for testing cannabis ingredients, including causing a reaction.   The method for examining cannabis components according to claim 16, wherein a final alcohol concentration during the antigen-antibody reaction is 7.5 wt% or less.   18. The solution according to claim 16 or 17, wherein the solution is a phosphate buffered saline (PBS-T) containing a surfactant, a phosphate buffered saline (PBS) containing no surfactant, or a pure water solution. The inspection method of the cannabis component as described.   The cannabis component inspection method according to any one of claims 16 to 18, wherein the alcohol is selected from the group consisting of methanol, ethanol and propanol.   The cannabis component testing method according to any one of claims 16 to 19, wherein the cannabis component is tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THC-A), or cannabinol (CBN).   21. The cannabis component inspection method according to any one of claims 16 to 20, wherein the target plant test piece includes any part selected from the group consisting of leaves, stems, roots, seeds, and petals.

Images