2.5∣Measurement of total IgE and specific For t 2 antibodies by
ELISA
The total IgE levels were measured using IgE mouse ELISA kit (Thermo
Fisher Scientific) according to the manufacturer’s instructions.
Ninety-six-well plates (Nunc) were coated overnight at 4℃ with 5 μg/mL
monoclonal antibody. All of the following steps were performed at room
temperature. After washing twice with PBS/0.05% Tween 20, the plates
were blocked with PBS plus 1% BSA for 2 hours and then incubated for 2
hours with prediluted serum (1:10) or 2-fold serial dilutions of mouse
IgE standards. After washing, detection antibody diluted 1:250 was added
and incubated for 2 hours. The plates were washed, and then
streptavidin-horseradish peroxidase conjugate diluted 1:400 was added.
After incubation for 30 minutes, tetramethylbenzidine (TMB) was added,
and the plate was incubated for 10 minutes. The reaction was stopped
with 1 M phosphoric acid, and absorbance was measured with an ELISA
reader (TECAN, Austria) at 450 nm.
The specific IgG1 and IgG2a to For t 2 were determined by in-house ELISA
with the required antibodies purchased from BD Pharmingen (San Jose, CA,
USA). Microtiter plates were coated with rFor t 2 for 2 hours at 37℃.
After washing with PBST, plates were blocked with 2% BSA for 2 hours at
room temperature. Sera were diluted (1:100 for IgG2a or 1:1000 for IgG1)
in PBST and incubated at room temperature for 2 hours. The plates were
washed and incubated with horseradish peroxidase-conjugated rabbit
anti-mouse IgG1 or IgG2a for 2 hours and developed by adding ABTS
solution (Sigma). The optical density was then analyzed on an ELISA
reader at 415 nm.