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.
Highlights
The most dangerous and poisonous mushroom is the death cap, Amanita phalloides
We further investigated the two most sensitive sera/antigen combinations: periodate oxidation (PERI)-AMA-keyhole hemocyanin (KLH)
A new newconjugation conjugation strategy, utilizing periodate oxidation (PERI) followed followed by amination, reductive strategy, utilizing periodate oxidation (PERI) followed by reductive amination, has employed for of for amination, has been been successfully successfully employed for the the development development of an an immunoassay immunoassay for amatoxin amatoxin has been successfully employed for the development of an immunoassay for amatoxin detection
Summary
The most dangerous and poisonous mushroom is the death cap, Amanita phalloides. A. phalloides are widely distributed around the world, predominantly thought to be an invasive species originating from Europe and it has been identified on every continent except Antarctica [1]. Amanita mushrooms contain several toxins, the bicyclic octapeptides known as amatoxins, and in particular α-amanitin (α-AMA) is considered the principle toxin (Figure 1). Impervious to heat, cold, or acid treatments. Amatoxins are potent inhibitors of the transcription process via inhibition of RNA polymerase II. A. phalloides contains variable amounts of toxins based
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