A new conjugation method used for the development of an immunoassay for amanitin, a deadly mushroom toxin

Authors: Bever, Candace; BARNYCH, BOGDAN - University Of California, Davis; Hnasko, Robert; Cheng, Luisa; Stanker, Larry

Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/21/2018
Publication Date: 6/28/2018
Citation: Bever, C.R., Barnych, B., Hnasko, R.M., Cheng, L.W., Stanker, L.H. 2018. A new conjugation method used for the development of an immunoassay for amanitin, a deadly mushroom toxin. Toxins. 10(7):265. https://doi.org/10.3390/toxins10070265.
DOI: https://doi.org/10.3390/toxins10070265

Interpretive Summary: One of the deadliest mushrooms is the death cap mushroom (Amanita phalloides) that contains toxins known as amatoxins. One amatoxin in particular is alpha-amanitin, which damages the liver and kidneys. In this paper, we have developed a tool, called an immunoassay, to detect these toxins. Immunoassays rely on an antibody molecule that binds to its target, in this case the toxin. To generate antibodies to alpha-amanitin, the toxin was chemically tethered to a larger protein molecule resulting in an immunogen that would generate antibodies to the toxin following immunizing of rabbits. Serum containing antibodies that bind alpha-amanitin were then isolated and used to develop this new immunoassay. This paper describes a new method for tethering alpha-amanitin to a larger carrier protein molecule. The resulting immunoassay detects amatoxins but not other similar toxins. The immunoassay detects as little as 1 ppb (1 ng/mL) toxin and was used to distinguish toxin containing death cap mushrooms from closely related mushrooms that do not contain this toxin. Thus, this test furthers our ability to monitor for toxin and improves the safety of the US food supply.

Technical Abstract: One of the deadliest mushrooms is the death cap mushroom (Amanita phalloides). The most toxic constituent is alpha-amanitin, a bicyclic octapeptide, which damages the liver and kidneys. To develop a new tool for detecting this toxin, polyclonal antibodies were developed and characterized. Both alpha- and ß-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 (LOD, ~ 1.0 ng/mL). The assays were characterized for their selectivity and were found to equally detect alpha-, ß-, and gamma-amanitin, and not cross-react with other toxins tested. Toxin detection in mushrooms was possible using a simple sample preparation method.