Opteia 555214

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.

Interferon gamma was detected using the “Mouse IFN-γ ELISA MAX” kit from Biolegend (#430805) in accordance with manufacturer’s instructions. Detection of anti-EBOV GP antibodies was performed by coating optical plates overnightwith soluble EBOV GP (50 ml/well at 10 mg/ml). Wells were washed 2x with PBST (0.015% Tween 20), blocked for 1 hour (PBS 2%BSA), washed 3x with PBST, and incubated overnight in 4°C with either a standard curve composed of fractionated mouse immunoglobulin (Immunore-agents #Mu-003-B) or serum samples. Following incubation wells were washed 4x with PBST and incubated with 50 mL rabbit anti-mouse IgG heavy and light chain-HRP (10 mg/ml Pierce #31457) for 1 hr at RT. Wells were then washed 5x with PBST, incubated with 50 mL of HRP substrate (BD OptEIA #555214). The reaction was stopped with 50 mL 2M H₂SO₄ and absorbance at 450nm was measured on Synergy H1 hybrid reader. Total concentration of anti-EBOV GP antibodies was quantified by comparison to the standard curve.