Characterization and purification of the mitochondrial antigens reactive with PBC sera

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We previously reported the presence of precipitating antibodies specific to one or more of three mitochondrial antigens (M-A, M-B, M-C) in sera from patients with primary biliary cirrhosis (PBC). It was also confirmed that antibodies to M-A and M-B are useful markers for the diagnosis of PBC. These mitochondrial antigens reactive with PBC sera were further characterized and purified in the present study.When a crude mitochondrial preparation was run on a DEAE Sephacel column using a 0.1-0.3M NaCl gradient, satisfactory resolution was not obtained among M-A, M-B, and M-C. However, when the same antigen preparation was eluted from a Sepharose 6 B column, peaks were obtained at molecular weight of 310kd, 240kd, and 155kd. These were found to correspond to M-C, M-A, and M-B, respectively, by counterimmunoelectrophoresis. Western blot analysis using a pool of the three Sepharose 6 B peaks as antigen showed that under reducing conditions, M-A antisera reacted with a 41kd band, M-B antisera with a 50kd band, and M-C antisera with a 31kd band. The pooled preparation was then purified and concentrated 1000× on a CNBr-activated Sepharose 4 B affinity column using anti-M-A and M-B IgG. Presently, additional characterization experiments are being conducted on these antigens in our laboratory.
日本臨床免疫学会の論文