Human IgE Antibody (Fc-2001)

Sandwich ELISA: Human IgE Antibody (Fc-2001) [NBP1-78601]

Sandwich ELISA analysis of human IgE was performed by coating wells ofa 96-well plate with100ul per well of a goat anti-human IgG (H+L) antibody diluted to a concentration of 1ug/ml incarbonate/bicarbonate buffer, and incubating overnight at4C. The plate was washed and blocked with Starting block T20 (TBS)Blocking Buffer for at least 30 minutes at roomtemperature, and then 100ul per well of human IgE, diluted in 1X PBS atconcentrations ranging from 1-1000ng/ml (left panel), and normal humanserum or serum from an asthma patient on anti-asthma prescriptionmedications (control serum, right panel), diluted in 1X PBS at dilutionsranging from 1:5 to 1:5000, was added to the plate and incubated for at least1 hour at room temperature. The plate was washed with 150ul per well of1X TBST, and 100ul of a human IgE monoclonal antibody was added to the wells at a concentration of 1ug/ml, and incubatedfor 1 hour at room temperature. The plate was washed and incubated with100ul per well of an HRP-conjugated goat anti-mouse IgG Fc secondaryantibody at a dilution of 1:8000 for at least 30 minutes atroom temperature. Detection was performed by adding 100ul per well of 1-Step Ultra TMB substrate and incubating for a few minutesat room temperature. The reactions were then stopped with TMB StopSolution. Absorbances were read on a spectrophotometerat 450-550nm.