Nunc Maxisorp plates were coated with capture antibodies (for cytokine ELISAs) or H. polygyrus lysate (for H. polygyrus-specific IgG1 ELISA) overnight. Following blocking, culture supernatants (cytokine ELISAs) or serum (IgG1 ELISA) were added, followed by incubation with cytokine-detection biotinylated antibodies or horseradish peroxidase (HRP)-conjugated anti-mouse IgG1 antibody. For cytokine ELISAs, streptavidin-conjugated horseradish peroxidase (HRP) was added. Substrate solution (BD OptEIA 555214) was added, reactions were stopped with 1 M H3PO4, and absorbance was read at OD450. For cytokine ELISAs, standard curves were generated with purified cytokines. For the IgG1 ELISA, serum was diluted between 10−2 and 10−8 and the last well with an OD450 above 0.100 represented the titer. The Mouse IL-4 and IL-5 ELISA Max Standard Sets (BioLegend), Mouse IL-13 DuoSet ELISA (R&D), and C57BL/6 Mouse Immunoglobulin Panel (SouthernBiotech) were used. For detection of GM-CSF, biotinylated αGM-CSF (BioLegend, MP1–31G6), unconjugated αGM-CSF (BioLegend, MP1–22E9), and murine GM-CSF (Peprotech) were used.
To generate H. polygyrus lysate, adult worms were washed in PBS and ground in 1 mL of PBS. Debris was pelleted by centrifugation at 16,000 g for 20 minutes at 4 °C. The supernatant was passed through a 0.2 μm filter and stored at −80 °C.
To generate H. polygyrus lysate, adult worms were washed in PBS and ground in 1 mL of PBS. Debris was pelleted by centrifugation at 16,000 g for 20 minutes at 4 °C. The supernatant was passed through a 0.2 μm filter and stored at −80 °C.