A 100-µg portion of the AEs was prepared with a ReadyPrep 2-D Cleanup kit (Bio-Rad
Laboratories) and used for two-dimensional gel electrophoresis (2-DE). First-dimensional
isoelectric focusing (IEF) and second dimensional SDS-PAGE were performed in accordance
with the manufacturer’s instructions (Bio-Rad Laboratories). Samples were applied to
rehydrated immobilized pH gradient (IPG) strips (7-cm long, pH range 3–10, Japan Bio-Rad
Laboratories) in a focusing tray. Then, the focusing IPG strips were run (Step 1: 250 V,
30 min; Step 2: 4,000 V, 60 min; Step 3: 4,000 V, 10,000 V-h) at 20°C under mineral oil.
After focusing, the strips were equilibrated, first in equilibration buffer I (Bio-Rad
Laboratories) for 20 min, and then in equilibration buffer II (Bio-Rad Laboratories) for
10 min. The strips were placed on top of the second-dimension gel (Bio-Rad Laboratories),
and electrophoresis was performed at 200 V for 30 min. The gel was visualized by staining
with CBB, and the other gel was transferred to NCS for Western blotting as described
above. Sera of individuals Nos. 93 and 195 working in the plant showing the highest OD in
IgG and IgA ELISA were used for detecting the recognized antigens, respectively.
Laboratories) and used for two-dimensional gel electrophoresis (2-DE). First-dimensional
isoelectric focusing (IEF) and second dimensional SDS-PAGE were performed in accordance
with the manufacturer’s instructions (Bio-Rad Laboratories). Samples were applied to
rehydrated immobilized pH gradient (IPG) strips (7-cm long, pH range 3–10, Japan Bio-Rad
Laboratories) in a focusing tray. Then, the focusing IPG strips were run (Step 1: 250 V,
30 min; Step 2: 4,000 V, 60 min; Step 3: 4,000 V, 10,000 V-h) at 20°C under mineral oil.
After focusing, the strips were equilibrated, first in equilibration buffer I (Bio-Rad
Laboratories) for 20 min, and then in equilibration buffer II (Bio-Rad Laboratories) for
10 min. The strips were placed on top of the second-dimension gel (Bio-Rad Laboratories),
and electrophoresis was performed at 200 V for 30 min. The gel was visualized by staining
with CBB, and the other gel was transferred to NCS for Western blotting as described
above. Sera of individuals Nos. 93 and 195 working in the plant showing the highest OD in
IgG and IgA ELISA were used for detecting the recognized antigens, respectively.