Measurement of Serum and Airway Immune Markers

Total serum IgE and IgG1, HDM-specific IgE and IgG1 levels were measured as previously described^{99 (link),100 (link)}. For measurement of HDM-specific IgE and IgG1 levels, wells were coated with 0.01% HDM (Greer Laboratories) overnight. Serum was diluted 1:5 for IgE and 1:2000 for IgG1. After 2 hours of incubation, plates were washed, and either horseradish peroxidase–conjugated anti-mouse IgG1 (X56; 1:1000; BD Biosciences PharMingen, San Jose, Calif) or biotin–anti-mouse IgE (R35-118; 1:250; PharMingen) was added for 1 hour, followed by an incubation with streptavidin–horseradish peroxidase (R&D DY998; 1:200) in the case of IgE. BALF cytokine were also quantified as previously described^{99 (link),100 (link)}. IL13 protein levels were measured using Mouse IL-13 DuoSet ELISA kit (R&D Systems, 62.5–4,000 pg/mL) per the manufacturer’s instructions. IL5 was measured using Mouse IL-5 ELISA MAX™ Standard kit (Biolegend, 7.8 pg/mL to 500 pg/mL) per the manufacturer’s instructions. IL33 protein levels in lung homogenates were measured using Mouse IL-33 DuoSet ELISA kit (R&D Systems, 15.6 pg/mL–1000 pg/mL) per the manufacturer’s instructions. The results were then presented in relation to total protein concentration for each sample.