After gel electrophoresis, proteins were transferred onto a nitrocellulose membrane. Membranes were blocked in 4% skimmed milk in TBST for 1 h, followed by serial incubation with EAO and normal serum samples (1:50) 4 °C overnight, a biotinylated goat anti-mouse whole IgG antibody (Amersham Biosciences, Freiburg, Germany; 1:10000), and finally, a streptavidin–HRP conjugate for 30 min (Amersham Biosciences). Bound antibodies were visualized using the ECL Plus Detection Reagent (Amersham Biosciences). Spots corresponding to the Western blot membrane were cut from the silver stained 2D gel for identification by MS. Peptidase protein digestion was performed as follows: A piece of a gel spot stained with silver was placed in a sampling tube, dried with a vacuum concentrator, rehydrated with a trypsin solution (10 μm/ml in 50 mM ammonium bicarbonate) and then placed in a small volume of 50 mM ammonium bicarbonate. After the incubation at 37 °C under vigorous shaking for 12–16 h, the supernatant solution was removed and stored for further use. The gel piece was successively extracted once with 50 μl and again with 25 μl of 5% trifluoroacetic acid/50% acetonitrile with shaking for 30 min each time. The supernatant and extracts were mixed and concentrated to a volume of 5–10 μl.