**1. Introduction**

There are thousands of reported mushroom poisonings occurring worldwide each year [1–5]. 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) and β-amanitin (β-AMA), and possibly γ-amanitin ( γ-AMA) (Figure 1). In mice, the LD50 for α-AMA is 0.1 mg kg−<sup>1</sup> [6] and, to humans, a dose of 0.3 mg kg−<sup>1</sup> is severely toxic [7]. AMAs are potent inhibitors of RNA polymerase II, with bioactivity resistant to heat, cold, or acid inactivation. 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<sup>−</sup><sup>1</sup> of α-AMA [8,10,11].

**Figure 1.** Chemical structures of the amatoxin variants examined in this paper, (**a**) molecular structure of amanitin, (**b**) R-group designations for each variant.

The most common method for the detection of AMAs extracted from mushrooms is liquid chromatography (LC), coupled with UV detection or mass spectrometry (MS) [8,12–14]. Although these methods are sensitive and provide a high resolution of individual analytes, they are time-consuming and require expensive, laboratory-based instrumentation and highly trained personnel to interpret the results. In contrast, immunoassays are faster, can be field portable, and require less sophisticated instrumentation. The only commercially available antibody-based assay for AMA detection for research purposes is the Bühlmann assay [15]. This assay relies on a polyclonal antibody (pAb), which is a limited supply. Once the supply of antibody is depleted, the assay will have to be reevaluated for sensitivity and selectivity using a newly produced pAb. Since monoclonal antibodies (mAbs) are produced by a hybridoma cell line derived from a single cell, they overcome this supply limitation and have little or no batch-to-batch variability. Similarly, recombinant antibodies can be produced in large quantities, while preserving the monoclonality of the binding domain. Assays utilizing mAbs or recombinant antibodies are thus more desirable for long-term consistency and can be scaled-up for test kit manufacture. To our knowledge, only a few mAbs to AMAs have been described, and only one has been used for analytical detection [16–18].

Regardless of the method used to detect the toxin, extraction of the AMA is required before identification. Over the years, the extraction procedure has been streamlined from 24 h [8,10,19] to one hour [12,14,16,20]. Most of these methods have utilized an extraction solution consisting of methanol, acid, and water. Results from a latter study using a one hour extraction reported levels of α-AMA to be 0.88–1.33 mg g<sup>−</sup><sup>1</sup> dry weight [12], while earlier studies using the 24 hour extraction reported comparable levels of 0.75–2.8 mg g<sup>−</sup><sup>1</sup> dry weight [8,10] for the same species. Despite potential differences in the ages of mushrooms studied, these consistencies across studies sugges<sup>t</sup> that extraction efficiency is not compromised with shortened extraction times. In addition, the historical methods use a combination of methanol, acid, and water to facilitate AMA extraction. Antibody-based immunoassays are often not compatible with large amounts of organic solvents or acidic solutions. Given the water solubility of AMAs, we hypothesized that a water-based AMA extraction would be sufficient for immunoassay detection.

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 mAbs. Then, we sought to use those mAbs to develop a sensitive and selective immunoassay for AMA detection from mushrooms. In this report, we describe and characterize novel anti-AMA mAbs and detail their performance in an indirect competitive inhibition enzyme-linked immunosorbent assay (cELISA). We compare the performance of this immunoassay for the detection of AMAs from

mushrooms using di fference extraction solutions. A sensitive detection assay for AMAs, combined with a rapid and simple toxin extraction method, would be a highly useful tool for the determination of AMA presence in wild mushrooms.
