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Toxins 2018, 10(7), 265; https://doi.org/10.3390/toxins10070265

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

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
2
Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California-Davis, Davis, CA 95616, USA
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
*
Author to whom correspondence should be addressed.
Received: 29 May 2018 / Revised: 19 June 2018 / Accepted: 21 June 2018 / Published: 28 June 2018
(This article belongs to the Special Issue Foodborne Toxins: Pathogenesis and Novel Control Measures)
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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. View Full-Text
Keywords: amatoxin; amanitin; death cap mushroom; Amanita phalloides; ELISA; immunoassay amatoxin; amanitin; death cap mushroom; Amanita phalloides; ELISA; immunoassay
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).
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Bever, C.S.; Barnych, B.; Hnasko, R.; Cheng, L.W.; Stanker, L.H. A New Conjugation Method Used for the Development of an Immunoassay for the Detection of Amanitin, a Deadly Mushroom Toxin. Toxins 2018, 10, 265.

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