2.4 Competitive ELISA
The inhibition ratios of β- and κ-CN against αs1-CN-specific IgE were
evaluated by competitive ELISA. The 96-well microtiter plates
(Nunc-ImmunoTM Plate II, Thermo Fisher Scientific) were coated with
αs1-CN (5 µg/mL in PBS) and incubated overnight at 4℃. After washing the
plates with PBS containing 0.05% Tween 20 (PBS-T), the plates were
blocked with PFBB containing 0.05% Tween 20 (PFBB-T) for 2 h at 4℃.
Patient sera (n = 39) were diluted 1:5 in PFBB-T and pre-incubated with
inhibitor (αs1-, β-, and κ-CN diluted in PBS at the final concentrations
of 0.001–100 μg/mL). The pre-incubated sera were added to each well and
incubated overnight at 4℃. After washing each well with PBS-T, bound
antibodies were detected with AP-conjugated goat anti-human IgE (diluted
1:1,000 in PFBB-T) for 1.5 h at 37℃. After washing each well with PBS-T
and PBS, PNPP substrate (Thermo Fisher Scientific) was added. The
reaction was stopped by the addition of 2 N NaOH and the absorbance was
measured at 405 nm. The inhibition ratio (%) was calculated using the
following formula:
Inhibition ratio (%) = 100 − (Inhibitor − NC) / (PC − NC) × 100
Inhibitor: absorbance of inhibitor solution; NC: Negative control,
absorbance of PFBB-T; PC: Positive control, absorbance of serum without
inhibitor