Abstract: Objective To improve a purifying method of Jo-1 antigen from rabbit thymus，which can be used for detection of anti-Jo-1 antibody by dot-blotting immunoassay (DB). Methods The rabbit thymus glands were cut into pieces, homogenized and extracted by PBS. Then the total protein was precipitated by acetone to get acetone powder (RTAP). The RTAP was solved in PBS and separated by an affinity column that was made by anti-Jo-1 IgG. Results 5~7g RTAP was obtained from 100g rabbit thymus glands. There was 19%~24% of protein in RTAP. Jo-1 antigen was enriched around 1900-folds through affinity chromatography, with 2.5% recovery of antigenic activity. In this preparation, there were several bands on SDS-PAGE, but only one band that was about 50 Ku, reacted with anti-Jo-1 antisera on immunoblotting. Dot-blotting also showed that the antigen only reacted with Jo-1 antisera, but not with any other antibodies related to rheumatic diseases. The purified Jo-1 antigen was not stable for long time, but the antigenic activity could maintain for a long time when there was MgCl2 in the solution. Conclusion Affinity chromatography was a simple and convenient method for purifying Jo-1 antigen from rabbit thymus. The antigen purified by affinity chromatography could meet the requirement for detecting Jo-1 antibody by dot-blotting. This method could be scaling up to produce Jo-1 antigen.