A Rapid Extraction Method Combined with a Monoclonal Antibody-Based Immunoassay for the Detection of Amatoxins.

Luisa W. Cheng,Larry H. Stanker,Candace S. Bever,Robert M. Hnasko

doi:10.3390/toxins11120724

Abstract

Amatoxins (AMAs) are lethal toxins found in a variety of mushroom species. Detection methods are needed to determine the occurrence of AMAs in mushroom species suspected in mushroom poisonings. In this manuscript, we report the generation of novel monoclonal antibodies (mAbs, AMA9G3 and AMA9C12) and the development of a competitive, enzyme-linked immunosorbent assay (cELISA) that is sensitive at 1 ng mL−1 and shows selectivity for α-amanitin (α-AMA) and γ-amanitin (γ-AMA), and less for β-amanitin (β-AMA). In order to decrease the overall time needed for analysis, the extraction procedure for mushrooms was also simplified. A rapid (1 min) extraction procedure of AMAs using solvents as simple as water alone was successfully demonstrated using Amanita mushrooms. Together, the extraction method and the mAb-based ELISA represent a simple and rapid method that readily detects AMAs extracted from mushroom samples.

Highlights

There are thousands of reported mushroom poisonings occurring worldwide each year [1,2,3,4,5]
The principle toxins responsible for the poisonings are the bicyclic octapeptides known as AMAs, most notably α-amanitin (α-AMA)
The aim of this study was to utilize our previously reported immunogen, a periodate-oxidized form of α-AMA conjugated to the keyhole limpet hemocyanin (PERI-AMA-KLH) [20], to generate mouse monoclonal antibodies (mAbs)

Summary

Introduction

There are thousands of reported mushroom poisonings occurring worldwide each year [1,2,3,4,5]. The most severe cases are from amatoxin (AMA)-containing mushrooms. AMA-containing mushrooms include a few species from the genera Amanita, Galerina, and Lepiota. The principle toxins responsible for the poisonings are the bicyclic octapeptides known as AMAs, most notably α-amanitin (α-AMA). The LD50 for α-AMA is 0.1 mg kg−1 [6] and, to humans, a dose of 0.3 mg kg−1 is severely toxic [7]. The typical distributions of α-AMA, β-AMA, and γ-AMA in a Death cap (Amanita phalloides) mushroom are approximately 43%, 43% and 14%, respectively [8,9]. A single dried mushroom typically contains around 1–2 mg g−1 of α-AMA [8,10,11]

Objectives

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Conclusion