Measurement of Human IL-4 Levels

The level of human IL-4 protein was measured using the Human IL-4 ELISA Set BD OptEIA™ (BD Biosciences) according to the manufacturer’s instructions. The protocol for specific IgG antibody detection has been previously described (41 (link)). Briefly, micro-wells of microtiter plates (Thermo Fisher Scientific) were coated with anti-human Igs (for total IgG detection), CH401MAP (for specific IgG detection), or BAL6MAP, a third-party antigen (N:NYYAGGFNSVRGFKDSTLGP) (1 μg/mL) diluted in carbonate buffer (pH 9.5), and the antigens were adsorbed to microtiter plates overnight at 4°C. The wells were washed with PBS-Tween (0.05% v/v) and blocked with 3% BSA-PBS at RT for 2 h. After three washes with PBS-Tween, 10-fold serial dilutions of mouse plasma were added to the wells and incubated for 2 h at RT. The plates were washed three times before adding biotin-conjugated mouse anti-human IgG mAb (BD Pharmingen, San Diego, USA) (1:3,000). After 2 h incubation at 37°C, the plates were washed 3 times, followed by the addition of streptavidin-horseradish peroxidase (1:50,000 v/v; BD Pharmingen). The plates were incubated for 1 h at RT, and unbound conjugates were removed by washing. Then, the EIA substrate kit solution (Bio-Rad Laboratories, Hercules, CA, USA) was added to each well. The reaction was stopped with 10% HCl, and the absorbance was measured at 450 nm.