Lateral flow immunoassay (LFIA) for the detection of lethal amatoxins from mushrooms

Abstract

The mushroom poison that causes the most deaths is the class of toxins known as amatoxins. Current methods to sensitively and selectively detect these toxins are limited by the need for expensive equipment, or they lack accuracy due to cross-reactivity with other chemicals found in mushrooms. In this work, we report the development of a competition-based lateral flow immunoassay (LFIA) for the rapid, portable, selective, and sensitive detection of amatoxins. Our assay clearly indicates the presence of 10 ng/mL of α-AMA or γ-AMA and the method including extraction and detection can be completed in approximately 10 minutes. The test can be easily read by eye and has a presumed shelf-life of at least 1 year. From testing 110 wild mushrooms, the LFIA identified 6 out of 6 species that were known to contain amatoxins. Other poisonous mushrooms known not to contain amatoxins tested negative by LFIA. This LFIA can be used to quickly identify amatoxin-containing mushrooms.

Rapid, Sensitive, and Accurate Point-of-Care Detection of Lethal Amatoxins in Urine

“…To ensure consistent interpretation of the line intensity by eye, the cut-off values determined in this study were used for the remainder of this study to determine the diagnostic accuracy of the test for urine analysis. The cut-off value for β-AMA when detected in urine resulted in a 20-fold more sensitive assay cut-off value than our previous standard curve developed in phosphate-buffered saline (PBS) [26]. There was no change in sensitivity for α-AMA or γ-AMA when using urine or PBS as the matrix [26].…”

“…The cut-off value for β-AMA when detected in urine resulted in a 20-fold more sensitive assay cut-off value than our previous standard curve developed in phosphate-buffered saline (PBS) [26]. There was no change in sensitivity for α-AMA or γ-AMA when using urine or PBS as the matrix [26]. To test whether the β-AMA sensitivity differences were possibly due to the pH of the matrix, we evaluated pH-adjusted PBS buffers ranging from 4.5 to 8 (Figure 3).…”

“…There was no change in sensitivity for α-AMA or γ-AMA when using urine or PBS as the matrix [26]. To test whether the β-AMA sensitivity differences were possibly due to the pH of the matrix, we evaluated pH-adjusted PBS buffers ranging from 4.5 to 8 (Figure 3).…”

“…Monoclonal antibody (AMA9G3), hapten-protein conjugate (LB-AMA-BSA), and full LFIA test strips were produced as described earlier [26,32,33]. The LFIA components are diagramed in Figure 6.…”

“…We have recently developed an LFIA for the detection of mushroom amatoxins [26]. As the sensitivity of the LFIA allows it to detect as little as 10 ng/mL, we hypothesized that this test would be useful for urine analysis in instances of mushroom poisonings.…”