A New Conjugation Method Used for the Development of an Immunoassay for the Detection of Amanitin, a Deadly Mushroom Toxin.

Toxins (Basel). 2018 Jun 28;10(7). pii: E265. doi: 10.3390/toxins10070265. Bever CS1, Barnych B2, Hnasko R3, Cheng LW4, Stanker LH5. Author information 1 Foodborne Toxin Detection and Prevention Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA. candace.bever@ars.usda.gov. 2 Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California-Davis, Davis, CA 95616, USA. bbarnych@ucdavis.edu. 3 Produce Safety and Microbiology Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA. robert.hnasko@ars.usda.gov. 4 Foodborne Toxin Detection and Prevention Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA. luisa.cheng@ars.usda.gov. 5 Foodborne Toxin Detection and Prevention Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA. larry.stanker@ars.usda.gov. Abstract One of the deadliest mushrooms is the death cap mushroom, Amanita phalloides. The most toxic constituent is α-amanitin, a bicyclic octapeptide, which damages the liver and kidneys. To develop a new tool for detecting this toxin, polyclonal antibodies were generated and characterized. Both α- and β-amanitin were coupled to carrier proteins through four different linking chemistries, one of which has never before been described. These conjugates were evaluated for their effectiveness in generating antibodies specific for the free toxin, as well as their utility in formatting heterogeneous assays with high sensitivity. Ultimately, these efforts yielded a newly described conjugation procedure utilizing periodate oxidation followed by reductive amination that successfully resulted in generating sensitive immunoassays (limit of detection (LOD), ~1.0 µg/L). The assays were characterized for their selectivity and were found to equally detect α-, β-, and γ-amanitin, and not cross-react with other toxins tested. Toxin detection in mushrooms was possible using a simple sample preparation method. This enzyme-linked immunosorbent assay (ELISA) is a simple and fast test, and readily detects amatoxins extracted from A. phalloides. KEYWORDS: Amanita phalloides; ELISA; amanitin; amatoxin; death cap mushroom; immunoassay PMID: 29958410 DOI: 10.3390/toxins10070265 Free full text