Biotinylated anti human ige

After electrophoresis, the separated proteins in the gel were transferred onto a nitrocellulose membrane (Millipore Co., Bedford, MA, USA), blocked with blocking buffer (3% bovine serum albumin in 1× PBS) overnight at 4°C, and incubated at room temperature (RT) for 4 hours with OVA⁺OM⁺-, OVA⁺OM⁻-, and OVA⁻OM⁻-pooled serum samples diluted to 1:20 in blocking buffer. For detecting an IgE-binding band, samples were incubated with biotinylated anti-human IgE (Vector Laboratories, Burlingame, CA, USA) diluted to 1:1,000 for 1 hour and then incubated with streptavidin-alkaline phosphatase (BD PharMingen, San Diego, CA, USA) diluted to 1:1,000 in blocking buffer for 15 minutes at RT. The membranes were developed using BCIP/NBT substrate (SIGMA FAST™ 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium; Sigma-Aldrich, St. Louis, MO, USA), and the development was terminated by adding distilled water. In all steps, the membranes were washed thrice with 0.05% Tween-20 in PBS (PBST, pH 7.0).19 (link)