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

#### **Candace S. Bever 1, Robert M. Hnasko 2, Luisa W. Cheng 1,\* and Larry H. Stanker 1**


Received: 22 November 2019; Accepted: 9 December 2019; Published: 11 December 2019

**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−<sup>1</sup> 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.

**Keywords:** amatoxins; amanitins; immunoassay; monoclonal antibodies; ELISA; death cap mushrooms

**Key Contribution:** Highly sensitive and selective monoclonal antibodies were developed to detect amatoxins. A rapid and simple extraction of amatoxins from mushroom specimens was demonstrated.
