Sandwich ELISA analysis of human IgE was performed by coating wells of a 96-well plate with100ul per well of a goat anti-human IgG (H+L) antibody diluted to a concentration of 1 µg/mL in carbonate/bicarbonate buffer, and incubating overnight at 4C. The plate was washed and blocked with StartingBlock T20 (TBS) Blocking Buffer for at least 30 minutes at room temperature, and then 100ul per well of human IgE, IgM, IgA, or IgG, diluted in carbonate/bicarbonate buffer at concentrations ranging from 1-1000ng/ml, was added to the plate and incubated for at least 1 hour at room temperature. The plate was washed with 150ul per well of 1X TBST, and 100ul per well of a human IgE monoclonal antibody 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 100ul per well of an HRP-conjugated goat anti-mouse IgG Fc secondary antibody at a dilution of 1:8000 for at least 30 minutes at room temperature. Detection was performed by adding 100ul per well of 1-Step Ultra TMB substrate and incubating for a few minutes at room temperature. The reactions were then stopped with TMB Stop Solution. Absorbances were read on a spectrophotometer at 450-550nm.