Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
National Institute of Health Sciences, 3-25-26, Tonomachi, Kawasaki-ku, Kawasaki 210-9501, Japan.
Anal Methods. 2021 Sep 16;13(35):3954-3962. doi: 10.1039/d1ay01157j.
A sensitive immunochemical method for identifying hallucinogenic mushrooms (magic mushrooms) is required for regulating their illicit use. We have previously generated a monoclonal antibody (mAb) that targets psilocin (Psi), the major psychoactive compound in hallucinogenic mushrooms, and developed an enzyme-linked immunosorbent assay (ELISA). However, this ELISA failed to achieve the expected low-picomole-range sensitivity, as a result of insufficient affinity of the mAb to Psi. It is recognized that haptenic antigens with a larger molecular mass tend to induce antibodies with higher affinities. Thus, we herein report a "derivatization-assisted ELISA," in which the "real analyte" Psi was determined as a "surrogate analyte," the -butyldimethylsilyl ether analog thereof (TBS/Psi) having a 1.6-fold greater molecular mass ( 318.53) than Psi. A novel mAb against TBS/Psi, prepared by immunizing mice with a TBS/Psi-albumin conjugate showed a 69-fold higher affinity to TBS/Psi residues ( = 3.6 × 10 M as IgG) than that of our previous mAb against Psi. This mAb consequently enabled a competitive ELISA for measuring TBS/Psi with the desired sensitivity: the dose-response curve midpoint (12.1 pmol per assay) was >100-fold lower than that of the previous ELISA for determining Psi. Extracts of dried mushroom powders were mixed with TBS triflate for 30 min at room temperature, converting Psi into TBS/Psi in approximately 50% yield. The reaction mixture was then subjected to an ELISA using the anti-TBS/Psi mAb to determine TBS/Psi. , a species of hallucinogenic mushrooms, gave rise to positive signals, indicating the presence of Psi therein in the expected quantity, while no detectable response was observed for four kinds of edible mushrooms available in the markets.
需要一种敏感的免疫化学方法来识别致幻蘑菇（迷幻蘑菇），以规范其非法使用。我们之前生成了一种针对致幻蘑菇中主要精神活性化合物裸盖菇素（Psi）的单克隆抗体（mAb），并开发了酶联免疫吸附测定（ELISA）。然而，由于 mAb 对 Psi 的亲和力不足，该 ELISA 未能达到预期的皮摩尔级灵敏度。人们认识到，具有更大分子量的半抗原抗原往往会诱导具有更高亲和力的抗体。因此，我们在此报告了一种“衍生化辅助 ELISA”，其中“真实分析物”Psi 被确定为“替代分析物”，其 - 丁基二甲基甲硅烷基醚类似物（TBS/Psi）的分子量比 Psi 大 1.6 倍（318.53）。通过用 TBS/Psi-白蛋白缀合物免疫小鼠制备的针对 TBS/Psi 的新型 mAb 对 TBS/Psi 残基的亲和力比我们之前针对 Psi 的 mAb 高 69 倍（=3.6×10^-9 M 作为 IgG）。该 mAb 因此能够用于测量 TBS/Psi 的竞争性 ELISA，具有所需的灵敏度：剂量反应曲线中点（每个测定 12.1 pmol）比以前用于测定 Psi 的 ELISA 低 100 多倍。干燥蘑菇粉提取物与 TBS 三氟甲磺酸酯在室温下混合 30 分钟，将 Psi 转化为 TBS/Psi，产率约为 50%。然后将反应混合物用抗 TBS/Psi mAb 进行 ELISA 测定 TBS/Psi。一种致幻蘑菇产生了阳性信号，表明其中存在预期数量的 Psi，而市场上四种可食用蘑菇则未观察到可检测到的反应。
