Immunochemical monitoring of psilocybin and psilocin to identify hallucinogenic mushrooms

Sep 1, 2020Journal of pharmaceutical and biomedical analysis

Using immune-based tests to detect hallucinogenic mushrooms by measuring psilocybin and psilocin

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Abstract

The enzyme-linked immunosorbent assays (ELISAs) developed can detect psilocybin and psilocin within ranges of approximately 0.20-20 μg/assay and 0.040-2.0 μg/assay, respectively.

Two independent monoclonal antibodies were generated specifically against psilocybin and psilocin.
The antibodies were produced using hybridoma technology involving mouse spleen cells.
Competitive ELISAs achieved a limit of detection of 0.14 μg/assay for psilocybin and 0.029 μg/assay for psilocin.
Cross-reactivity in the assays was minimal, with psilocybin and psilocin showing only 2.8% and less than 0.5% cross-reactivity, respectively.
Psilocybin and psilocin were quantified in dried Psilocybe cubensis mushrooms at 0.39% and 0.32% (w/w), respectively.

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Development of rapid and reliable immunochemical methods for monitoring psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine; Pyb) and psilocin (dephosphorylated metabolite; Psi), the psychoactive compounds contained within hallucinogenic mushrooms (magic mushrooms), is desirable in order to identify these mushrooms and regulate their illicit use. Because no antibody was publicly available for this purpose, we generated two independent monoclonal antibodies (mAbs) against Pyb or Psi, and then developed enzyme-linked immunosorbent assays (ELISAs) by using them. To generate the specific antibodies, novel immunogenic conjugates were prepared by linking Pyb or Psi molecules to carrier proteins by modifying their 2-(N,N-dimethylamino)ethyl side chains. Spleen cells from mice immunized with these conjugates were fused with P3/NS1/1-Ag4-1 myeloma cells, and hybridoma clones secreting anti-Pyb and anti-Psi mAbs were established. These mAbs were characterized for their biochemical features and then applied to competitive ELISAs, which used microplates coated with Pyb or Psi linked with albumin. These ELISAs enabled the determination of Pyb or Psi with measurable ranges of ca. 0.20-20 or 0.040-2.0 μg/assay (limit of detection was 0.14 or 0.029 μg/assay), respectively. The related tryptamines were satisfactorily discriminated as exemplified by the cross-reactivity of the ELISA to determine Pyb (or Psi) with Psi (or Pyb) that were found to be 2.8 % (or <0.5 %), respectively. The Pyb and Psi contents in a dried powder of the hallucinogenic mushroom, Psilocybe cubensis, were determined to be 0.39 and 0.32 (w/w)%, respectively. The ELISAs developed using the current mAbs are promising tools for identifying illegal hallucinogenic mushrooms.

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Immunochemical monitoring of psilocybin and psilocin to identify hallucinogenic mushrooms

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