Aluminum hydroxide gel adjuvant

FA induction was performed as previously described [2 (link),10 (link),11 (link)]. Briefly, 5-week-old male BALB/c mice were sensitized twice, with an intervening two-week interval, by intraperitoneal injection of 50 μg of OVA (fraction V; Sigma-Aldrich, St. Louis, MO, USA) in the presence of an aluminum hydroxide gel adjuvant (Thermo scientific, Rockford, IL, USA). Two weeks later, the mice were repeatedly given 50 mg of OVA every other day. Diarrhea was assessed by visually monitoring the mice for up to 1 h following the oral OVA challenge. The profuse liquid stool was detected as allergic diarrhea, and the diarrhea-presenting mice were considered to be FA mice. The occurrence of allergic diarrhea reached about 80.0% after the 6th OVA challenge. Tissue samples were collected 1 h after the 6th oral OVA challenge.

Polyclonal monospecific antibodies against rCsGSTo1 and 2 were generated in 6-week-old female BALB/c mice. The recombinant proteins (100 μg) mixed with 2% aluminum hydroxide gel adjuvant (Thermo Fisher Scientific) were subcutaneously immunized 3 times with 2-week intervals. One week later, a final booster was administered through intravenous route (10 μg). Blood was collected by heart puncture. IgG fractions were purified by a Protein G affinity column.

The Institutional Animal Care and Use Committee of China Medical University approved the experimental animal protocol of the study. All animals were maintained and treated in accordance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press. Female BALB/c mice at 6–8 weeks of age were obtained from the National Laboratory Animal Center in Taiwan. Experimental mice were sensitized with intraperitoneal injection of 4 μg house dust mite (HDM, Indoor Biotechnologies Ltd, Cardiff, UK) mixed with 40 μg aluminum hydroxide gel adjuvant (Invitrogen, San Diego, CA) on days 0, 7, and 14, and received intranasal challenge with 4 μg HDM from days 22 to 26 (Kim et al., 2014 (link)).