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WO2024129040A1 - Test sur carte pour la molécule de psilocybine hallucinogène qui permet le diagnostic de champignons toxiques et de toxicités de champignons - Google Patents

Test sur carte pour la molécule de psilocybine hallucinogène qui permet le diagnostic de champignons toxiques et de toxicités de champignons Download PDF

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Publication number
WO2024129040A1
WO2024129040A1 PCT/TR2023/051529 TR2023051529W WO2024129040A1 WO 2024129040 A1 WO2024129040 A1 WO 2024129040A1 TR 2023051529 W TR2023051529 W TR 2023051529W WO 2024129040 A1 WO2024129040 A1 WO 2024129040A1
Authority
WO
WIPO (PCT)
Prior art keywords
analyte
psilocybin
test
molecule
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/TR2023/051529
Other languages
English (en)
Inventor
Ilgaz AKATA
Ertugrul KAYA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
TC Ankara Universitesi
Original Assignee
TC Ankara Universitesi
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from TR2022/019556 external-priority patent/TR2022019556A2/tr
Application filed by TC Ankara Universitesi filed Critical TC Ankara Universitesi
Publication of WO2024129040A1 publication Critical patent/WO2024129040A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • G01N33/942—Serotonin, i.e. 5-hydroxy-tryptamine

Definitions

  • the invention falls within the technical field of biochemistry and relates to an immunochromatographic card test for the diagnosis of the psilocybin molecule, which allows the diagnosis of toxic mushrooms and mushroom toxicities and has a hallucinogenic effect on the brain.
  • the invention essentially relates to a card test that allows the diagnosis of toxic mushrooms and mushroom toxicity and is more economical than current applications and requires less expertise and shorter periods of time, for the diagnosis of the psilocybin molecule, which has a hallucinogenic effect on the brain.
  • the invention relates to a method of performing the immunochromatographic card test for the diagnosis of the psilocybin molecule, which allows the diagnosis of toxic mushrooms and mushroom toxicities and has a hallucinogenic effect on the brain.
  • the hallucinogenic effects of psilocybin-containing mushrooms have been known for 2000 years. In particular, it is known that the Mayans and Aztecs, the civilizations of the South American region, used these mushrooms. Its chemical formula is N,N-dimethyl-4- phosphoryloxytryptamine.
  • the Psilocybin molecule is known to be present in the fungus species Psilocybe cubensis, Psilocybe argentipes, Psilocybe mexicana, Psilocybe bohemica, Panaeolus cyanoscens, Panaeolus foenisecii, Gymnophilus purpuratus, Stropharia semiglobata. Apart from these species, this molecule can also be found in some other fungus species. As is known in the art, it is known to cause LSD-type hallucinations. Its chemical structure is provided as Formula 1.
  • Toxicities are especially seen in autumn and spring, during which mushrooms grow intensively. Despite all the developments in modern medicine, no definitively effective medicine has been found for the treatment of certain mushroom toxicities.
  • the main toxins present in mushrooms are Cyclopeptides (Amanitins, phallotoxins, virotoxins) (Hepatotoxic); Giromitrin (Monomethylhydrazine) (NephroHepato-Neurotoxic); Orellanin (Nephrotoxic); Muscarine (Muscarinic effect, Autonomic Nervous system); Ibotenic acid and Muskimol (Hallucinogenic); Coprin (Disulfiram-like effect) and Psilocybin (Hallucinogenic).
  • the invention relates to a card test for the detection of toxicity and/or hallucinogenic cases caused by a toxic substance, psilocybin molecule, contained in the mushroom.
  • the invention offers a test method which is less costly and does not require well- equipped equipment, therefore eliminates the need for qualified personnel and provides rapid results by an antibody-antigen interaction using an antibody suitable for the detection of toxicity and/or hallucinogenic cases caused by a toxic substance, psilocybin molecule, contained in the mushroom.
  • the invention relates to an immunochromatographic card test for detecting the presence of psilocybin molecules, which allows the diagnosis of toxic mushrooms and mushroom toxicities, which test comprises o at least one test line containing psilocybin molecules immobilised to the membrane, o an analyte for the determination of antibody-antigen interaction, o a conjugate pad to which monoclonal antibodies specific to said analyte are added, wherein said antibodies include mouse immunoglobulin G monoclonal antibodies, and which is based on a principle of single line (positive)-double line (negative) by moving towards the test line after the interaction of the analyte and antibodies and then competing with the psilocybin molecule immobilised to the membrane.
  • said membrane is based on nitrocellulose.
  • the invention also relates to an in-vitro method for detecting the presence of the psilocybin molecule, which allows the diagnosis of toxic mushrooms and mushroom toxicities.
  • the method of the invention includes the following process steps:
  • said monoclonal antibody includes mouse immunoglobulin G monoclonal antibodies
  • test line if the test line is single, then analyzing the test as positive.
  • the invention also relates to an immunochromatographic card test for detecting the presence of psilocybin, which allows the diagnosis of toxic mushrooms and mushroom toxicities, which comprises
  • said colored monoclonal antibody includes mouse immunoglobulin G monoclonal antibodies
  • test line containing specific monoclonal antibodies and which is based on the principle that the analyte selectively binds to the specific colored antibody thereof on the conjugate pad, a complex of the analyte-antibody-color molecule moves towards the test line together with the analyte, and then a visible colored line is formed in the test line area.
  • the invention also relates to an in-vitro method for detecting the presence of the psilocybin molecule, which allows the diagnosis of toxic mushrooms and mushroom toxicities.
  • the method of the invention includes the following process steps:
  • said colored monoclonal antibody includes mouse immunoglobulin G monoclonal antibodies
  • the subject matter of the invention falls within the technical field of biochemistry and relates to a card test for the diagnosis of the psilocybin molecule, which allows the diagnosis of toxic mushrooms and mushroom toxicities and has a hallucinogenic effect on the brain, but is described with examples only for a better understanding of the subject in a non-limiting sense.
  • the basic working principle of the card test of the invention is based on an immunochromatographic method.
  • the test card of the invention includes at least one sample pad, a conjugate pad, at least one membrane, at least one test line, at least one control line, at least one absorbent pad, and/or at least one plastic base.
  • the elements given here are the basic components of immunochromatographic tests, and changes may occur in the number or functions of the elements for a high-efficiency diagnosis due to the advancement of the technology. As mentioned, the present inventors may keep up with the changes in immunochromatographic cards and make changes in the number and functions of the components in the card test.
  • the protection scope of the invention is independent of the elements included in the card test based on the immunochromatographic test method.
  • the protection scope of the invention is essentially based on the fact that there is no need for less costly and well-equipped equipment and thus no qualified personnel is required and the rapid results are obtained, by means of the immunochromatographic method based on an antibody-antigen interaction for the diagnosis of the psilocybin molecule, which previously required the presence of the expensive devices, qualified personnel and well-equipped laboratories.
  • the sample pad in the card test of the invention is used to drop the liquid material to be analyzed and keeps the dropped liquid.
  • the liquid material to be analyzed may be, but is not limited to, the juice of the mushroom, or the serum of the poisoned patient.
  • the conjugate pad in the card test of the invention contains psilocybin-specific antibodies marked and colored with gold nanoparticles.
  • the sample liquid passes over this pad and drags the colored antibodies on the pad.
  • Psilocybin-specific antibodies are specific to this invention and are produced by the inventors.
  • the psilocybin molecules are injected into the experimental mice, the mice are treated with a series of immunogens, and after a while, the B lymphocytes in the spleens of the mice are collected and the cells that produce antibodies specific to psilocybin are isolated.
  • the monoclonal psilocybin antibodies produced by these cells are used as reported in the invention. Consequently, specific antibodies to the psilocybin molecule have been produced specifically for this invention and are psilocybin-specific mouse IgG type monoclonal antibodies.
  • the membrane in the card test of the invention is used to move the liquid sample towards the test line by the effect of capillary flow. Different membranes may be used for this purpose, but nitrocellulose membrane is often used.
  • the sample test line contains membrane-immobilised psilocybin molecules if it is to be produced in a competitive model. The quantity of these molecules is adjusted according to the desired threshold value during R&D studies. If the test is to be produced in a sandwich model, then the psilocybin-specific antibodies are immobilised to the test line.
  • the sample should move down to the control line in the card test of the invention, otherwise the test will be considered invalid.
  • the anti-antibodies are immobilised on the membrane on this line.
  • the absorbent pad in the card test of the invention is placed at the endmost point and is responsible for retaining the excess of the liquid sample. When a large amount of liquid sample is dropped into the test, it accumulates in the pad and contributes to the correct functioning of the test.
  • the card test of the present invention may function with two types of working principles. Accordingly,
  • the operation method of the card test is as follows:
  • the analyte in the sample selectively binds to the colored specific antibody on the conjugate pad, and the complex of the analyte-antibody-color molecule moves towards the test line along with the sample.
  • the analyte in the complex arrived at the test line together with the sample binds to the antibodies immobilised to the test line and causes a colored visible line in the test line area. As a color will appear on the control line in every case where the test is valid, a double line will be seen in the observation window as a positive result of the sandwich model. If the analyte sought is not found, the result of the test is expected to be negative. As there is no analyte in the sample, there is no binding to the colored antibody on the conjugate pad.
  • the operation method of the card test is as follows:
  • a specific monoclonal antibody is added to the analyte colored by binding a color molecule on the conjugate pad. These antibodies are not immobile, and these colored antibodies are draft towards the test line with the incoming liquid sample.
  • the analyte to be analyzed is immobilised to the test line area.
  • the result is positive if a single line is seen in the observation window, and the result is negative if a double line is observed. Due to this feature, the models described give contradictory results, and care should be taken during a practical application. If the analyte sought is found in the sample, the result of the test is expected to be negative (double line).
  • the technical innovation for which the invention claims an essential protection and provides for the relevant technical field is the introduction of the use of the immunochromatographic methods based on an antibody-antigen interaction for the diagnosis of the hallucinogenic psilocybin molecule contained in the mushroom.
  • specially produced monoclonal antibody molecules are used as antibodies that will specifically interact with the psilocybin molecule as an antigen.
  • the antibodyantigen complex is a highly selective binding, the molecule desired to be analyzed may be successfully detected.
  • Said antibody molecule is a special molecule which selectively recognizes the analyte. Due to a high antibody-antigen selectivity ratio, the diagnostic reliability of the card tests of the invention is quite high.
  • the card test which makes this interaction possible thanks to the antibody-antigen selective relationship, will provide reliable test results by providing a high diagnosis rate without requiring an equipped laboratory and qualified personnel.
  • the card test of the invention has a simple structure without requiring high equipment for the diagnosis of the psilocybin molecule in mushrooms in the present art. Therefore, as the production costs will be low, sales will also be economical. In this way, it will be possible to obtain a diagnostic product at affordable prices that many people associated with the mushroom habitat may easily obtain.
  • the card test of the present invention allows the test results to be obtained in a short time.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un test sur carte qui permet le diagnostic de champignons toxiques et de toxicités de champignons et est plus économique que les applications actuelles et nécessite moins d'expertise et des temps plus courts, pour le diagnostic de la molécule de psilocybine, qui a un effet hallucinogène sur le cerveau.
PCT/TR2023/051529 2022-12-16 2023-12-12 Test sur carte pour la molécule de psilocybine hallucinogène qui permet le diagnostic de champignons toxiques et de toxicités de champignons Ceased WO2024129040A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
TR2022019556 2022-12-16
TR2022/019556 TR2022019556A2 (tr) 2022-12-16 Zehi̇rli̇ mantarlarin ve mantar zehi̇rlenmeleri̇ni̇n teşhi̇si̇ne olanak sağlayan halusi̇nojeni̇k etki̇ye sahi̇p psi̇losi̇bi̇n molekülü i̇çi̇n bi̇r kart testi̇

Publications (1)

Publication Number Publication Date
WO2024129040A1 true WO2024129040A1 (fr) 2024-06-20

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PCT/TR2023/051529 Ceased WO2024129040A1 (fr) 2022-12-16 2023-12-12 Test sur carte pour la molécule de psilocybine hallucinogène qui permet le diagnostic de champignons toxiques et de toxicités de champignons

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020210658A1 (fr) * 2019-04-10 2020-10-15 Promega Corporation Compositions et procédés pour la détection d'analytes au moyen d'une bioluminescence

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020210658A1 (fr) * 2019-04-10 2020-10-15 Promega Corporation Compositions et procédés pour la détection d'analytes au moyen d'une bioluminescence

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BEVER CANDACE S., ADAMS CATHARINE A., HNASKO ROBERT M., CHENG LUISA W., STANKER LARRY H.: "Lateral flow immunoassay (LFIA) for the detection of lethal amatoxins from mushrooms", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, US, vol. 15, no. 4, 17 April 2020 (2020-04-17), US , pages 1 - 18, XP093184367, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0231781 *
SUKUMARAN A ET AL.: "Development and Troubleshooting in Lateral Flow Immunochromatography Assays", INDIAN J CLIN BIOCHEM., vol. 36, no. 2, 17 April 2020 (2020-04-17), pages 208 - 212, XP037395089, [retrieved on 20210400], DOI: 10.1007/s12291-020-00887-5 *
ZHU J ET AL.: "Production of highly sensitive monoclonal antibody and development of lateral flow assays for phallotoxin detection in urine", ANAL BIOANAL CHEM., vol. 413, no. 20, 8 July 2021 (2021-07-08), pages 4979 - 4987, XP037551337, [retrieved on 20210800], DOI: 10.1007/ s 00216-021 -03457-7 *

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