The reaction was stopped after 15 min by the addition of 5% SDS. Reactivity was measured using a Wallac spectrophotometer at a wavelength of 405 nm. Known positive and negative sera were included with each assay. For immunoblotting, samples were suspended in 250 ml of sample buffer (125 M Tris -HCl (pH6.8) containing 4% SDS, 20% glycerol and 5% 2-ME), boiled for 5 min, and resolved by SDS-PAGE using 1.5 mm-thick slab gels with a 4.75% stacking gel and a 10% separating gel. Separated proteins were transferred electrophoretically to nitrocellulose filters purchased from Micron Separations (Westboro, MA). After transfer, nitrocellulose filters were cut into strips, blocked with 3% milk in PBS for 1 h at RT and probed by incubation for 1 h with mouse sera diluted at 1:200. After washing with PBS/Tween, strips were incubated for an additional hour with HRP-labeled goat anti-mouse or anti-human polyvalent immunoglobulin (1: 2000). Strips were washed and visualized with enhanced chemiluminescent substrate purchased from Pierce (Rockford, IL). Known AMA positive PBC sera and control negative sera were used throughout these studies as controls. Immunohistochemical staining of Hep-2 cells were performed using Hep-2 cell slides purchased from Antibodies Inc. (Davis, CA). Slides were incubated at RT for 1 h with SJL/J mice sera, human PBC sera or control sera diluted at 1:40. After incubation, slides were washed in PBS for 10 min. The reaction was followed by a 30 min incubation with FITC-conjugated goat anti-mouse or anti-human IgG diluted in PBS. Once again, known positive and negative sera were used throughout.