Pathogen-Specific Immunoglobulin Binding Assay

To assess whether infected and survivor fish had generated pathogen-specific immunoglobulins, we measured the capacity of IgT, IgM and IgD from serum, gill mucus or tissue (gill, spleen and head kidney) explant supernatants to bind to Ich using a pull-down assay previously described by us9 (link). In short, parasites (∼100 tomonts) were preincubated with a solution of 0.5% BSA in PBS (pH 7.2) for 2 h at 4 °C. Thereafter, parasites were incubated with diluted gill mucus or serum or tissue (gill, head kidney and spleen) explant supernatants from infected, survivor or control fish for 2 h at 4 °C in a 300 μl volume. Dilutions were made with PBS containing 0.5% BSA (pH 7.2). After incubation, the tomonts were washed with PBS, and bound proteins were eluted with Laemmli Sample Buffer (Bio-Rad) and boiled for 5 min at 95 °C. The eluted material was resolved on 4–15% SDS–PAGE Ready Gel under non-reducing conditions, and the presence of IgT, IgM or IgD was detected by western blotting using the anti-trout IgT, IgM or IgD antibodies as described above. Original images of the western blot analyses are shown in Supplementary Fig. 11.