Western blot analysis of human IgE was performed by loading 0.5 µg of purified human IgE, IgM, IgA and IgG per well and 10 µL of PageRuler Prestained Protein Ladder (Product # 26616) per well onto a Novex® 4-20% Tris-glycine polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288), and blots were blocked with 5% milk in TBST for at least 1 hour at room temperature. Human IgE was detected at ~75 kD using a human IgE monoclonal antibody (Product # MA5-14704) at a dilution of 1:1000 in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse light chain secondary antibody at a dilution of 1:40,000 for at least 30 minutes at room temperature (left blot). To control for protein loading, a duplicate blot was probed with a mixture of goat anti-human IgG, IgM, and IgA (Product # 31128) and goat anti-human IgE (Product # A18801) antibodies at concentrations of 1 µg/mL, followed by a mouse anti-goat IgG-HRP secondary antibody (Product # 31400) at a dilution of 1:8000 (right blot). Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).

Sandwich ELISA analysis of human IgE was performed by coating wells of a 96-well plate with100 µL per well of a goat anti-human IgG (H+L) antibody (Product # 31119) diluted to a concentration of 1 µg/mL in carbonate/bicarbonate buffer (Product # 28382), and incubating overnight at 4C. The plate was washed and blocked with StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for at least 30 minutes at room temperature, and then 100 µL per well of human IgE, diluted in 1X PBS at concentrations ranging from 1-1000 ng/mL (left panel), and normal human serum or serum from an asthma patient on anti-asthma prescription medications (control serum, right panel), diluted in 1X PBS at dilutions ranging from 1:5 to 1:5000, was added to the plate and incubated for at least 1 hour at room temperature. The plate was washed with 150 µL per well of 1X TBST, and 100 µL of a human IgE monoclonal antibody (Product # MA5-14704) was added to the wells at a concentration of 1 µg/mL, and incubated for 1 hour at room temperature. The plate was washed and incubated with 100 µL per well of an HRP-conjugated goat anti-mouse IgG Fc secondary antibody (Product # 31439) at a dilution of 1:8000 for at least 30 minutes at room temp. Detection was performed by adding 100 µL per well of 1-Step Ultra TMB substrate (Product # 34028) and incubating for a few min at room temperature. The reactions were stopped with TMB Stop Solution (Product # N600). Absorbances were read on a spectrophotometer at 450-550 nm.