Fig 5. Detection of epsilon toxin and prototoxin using an immunochromatographic test.

(1) Serial dilutions of epsilon toxin and prototoxin were prepared in immunochromatographic (ICT) buffer (A), buffered tap water (B), milk (C), buffered and clarified ovine jejunum content from sheep 111136 (D) or buffered human plasma (E) before detection with the lateral flow immunoassay developed using PεTX5 mAb (test line) and a goat anti-mouse immunoglobulin antibody (control line) as capture antibodies, and colloidal gold-labeled PεTX7 mAb as tracer. Milk, tap water and human plasma were previously buffered, i.e. addition of one-tenth volume of 10× ICT buffer (1× final ICT buffer in all different matrices). Clarified intestinal content was previously diluted 2-fold in a final concentration of 25% fetal calf serum and 1× ICT buffer, before being spiked. (2) Serial dilutions of epsilon toxin were prepared in commercial Bio-X buffer (test strips kit BIO K 176, Bio-X) (A), in milk previously half-diluted in commercial Bio-X buffer (C), in ovine jejunum content from sheep 111136 prepared following the instructions provided in the kit (D) or in human plasma previously half-diluted in commercial Bio-X buffer (E). These epsilon toxin preparations were analyzed using the commercial test strips BIO K 176 (Bio-X) following the instructions provided in the kit.