The sampling method for household dust collection was based on the protocol of Tsutsui et al. with slight modifications 16 . Household dust was obtained from the living rooms and bedrooms where each patient spent the most time via a domestic vacuum cleaner with a 40‐µm pore‐size sampling filter (Dustream Collector, Indoor Biotechnologies, Inc., Charlottesville, VA).
Approximately 100 mg of dust from each sample was added to 4 ml of PBS with Tween 20 and was shaken for 2 h at room temperature. After centrifugation for 5 min at 500 × g, the supernatant solution was filtered through a 40 µm filter (Cell Strainer, BD Biosciences, San Jose, CA), then filtered through a 0.45 µm syringe filter (Stertile Acrodisc® Syringe Filters with Supor® Membrane, PALL Life Sciences, Ann Arbor, MI). The dust extract solutions were stored frozen at −20°C until analysis.
Monoclonal and polyclonal antibodies against pigeon dropping extract were generated according to the methods of previous studies15 , 16 . A sandwich ELISA with monoclonal and polyclonal antibodies was used to measure the AAA as described previously 16 . To increase the sensitivity of avian antigen detection, we used the ELAST amplification system (ELAST®, PerkinElmer Life Sciences, Inc., Waltham, MA) per the manufacturer's instructions. The AAA is expressed in micrograms of pigeon dropping extract per 1 gram of dust.
Approximately 100 mg of dust from each sample was added to 4 ml of PBS with Tween 20 and was shaken for 2 h at room temperature. After centrifugation for 5 min at 500 × g, the supernatant solution was filtered through a 40 µm filter (Cell Strainer, BD Biosciences, San Jose, CA), then filtered through a 0.45 µm syringe filter (Stertile Acrodisc® Syringe Filters with Supor® Membrane, PALL Life Sciences, Ann Arbor, MI). The dust extract solutions were stored frozen at −20°C until analysis.
Monoclonal and polyclonal antibodies against pigeon dropping extract were generated according to the methods of previous studies