Fig 1.
Chemical structures of the amatoxin variants examined in this paper.
(a) molecular structure of amanitin. (b) R-group designations for each variant.
Fig 2.
Depictions of the test strips used in this study.
(a) Schematic diagram of the lateral flow strip along with a diagram of the reagents on the control line (CL) and test line (TL). (b) A view of the strips when used in a cassette. The left cassette is an example of a sample without amatoxins (negative) and the right cassette is an example of a sample with amatoxins (positive). (i) sample pad, (ii) conjugate pad, (iii) nitrocellulose membrane, (iv) wicking pad, and the arrow indicates the flow direction.
Fig 3.
Visual representation of the lateral flow immunoassay (LFIA) half strips.
Test (T) line coating antigens were (a) LB-AMA BSA and (b) PERI-AMA-BSA immobilized onto six different nitrocellulose membrane types: (1) MDI 150, (2) FF120, (3) FF80, (4) MDI 90, (5) CN95, and (6) CN140. (*) designates the preferred membrane used in the remaining experiments.
Fig 4.
Standard calibration curves of (a) α-amanitin, (b) β-amanitin, and (c) γ-amanitin determined by lateral flow immunoassay (LFIA). The images on the left (a-c) are the test strips. The graphs to the right (d-f) are the test line pixel values (red circles) and visual score values (blue triangles) from the corresponding image (a-c) expressed as a mean ± standard error, for three separate strips.
Table 1.
LFIA test results for pure chemical toxin standards of chemicals from associated mushrooms or are other peptide toxins.
Fig 5.
Shelf-life testing of the LFIA stored at (a) 45 °C and (b) 55 °C.
Minimal loss of signal was observed over the course of 25 days for those tested at 55 °C and over the course of 44 days for those tested at 45 °C. The LFIA performance was tested using 3 different concentrations of α-AMA (0, 1, and 10 ng/mL) in PBS.
Table 2.
Species names and UC Herbarium codes for wild mushrooms sampled in this study and mentioned in Fig 6.
A ‘Y” in the “ITS” (internal transcribed spacer) column indicates that the ITS region was sequenced and assigned a species based on the highest percent match when BLAST searched in the NCBI database. Bold text indicates those species that are known to contain amatoxins. Those marked with an * were subjected to chemical analysis by LC-MS.
Fig 6.
LFIA results from mushroom extracts.
The mushrooms are as follows: 1) Amanita augusta, 2) A. bisporigera*, 3) A. calyptratoides, 4) A. constricta, 5) A. gemmata, 6) A. magniverrucata, 7) A. marmorata#, 8) A. muscaria, 9) A. novinupta, 10) A. ocreata*, 11) A. pantherina, 12) A. phalloides*, 13) A. protecta, 14) A. velosa, 15) Agaricus californicus, 16) Ag. xanthodermus, 17) Boletus edulis, 18) Cantharellus californicus, 19) Galerina marginata*, 20) G. sideroides, 21) Lepiota subincarnata*, 22) Pholiotina utricystidiata, and 23) Volvariella volvacea. Those marked with an * were confirmed by LC-MS analysis to contain α-AMA, and the sample marked with a # was confirmed by LC-MS analysis to contain phallotoxins. 74 additional mushroom species tested were negative by LFIA. Names of all mushrooms tested are provided in Table 2.