Antimitochondrial antibodies were assayed using a combination of ELISA, immunoblotting and immunohistochemical staining of HEp-2 cells. For ELISA, microtiter plates were coated with 10 mg/ml PDC-E2 suspended in carbonate buffer (pH 9.6) and incubated overnight at 48C. After washing three times with 0.05% Tween-20 in PBS (PBS/Tween), plates were blocked with 3% milk in PBS for 1 h at room temperature (RT). Diluted mouse sera (1:200) was distributed into individual wells and incubated for 1 h at RT. After washing, the plates were incubated with 0.1 ml of HRP-conjugated goat anti-mouse immunoglobulin (Zymed, South San Francisco, CA) for 1 h. Reactivity was visualized by addition of 40 mM 2,2 0azinobis (3-ethylbenzthiazoline-6-sulfonic acid) dissolved in 0.05 M citric acid buffer (pH 5) containing 0.5 M H 2 O 2 .