Abstract: Objective　To establish a complement-meditated indirect immunofluorescence method（two-step method） for detecting anti-perinuclear factor（APF） and apply it primarily. Methods　The epithelial cells of oral cheek from non-smoking healthy and non-connective tissue disease subjects were used as substrate. The diluted serum，complement and fluorescent label anticomplement C_3C were added. The nuclear fast red-glycerol was performed. By microscopy，the sizes with round or oval homogeneous fluorescent granules in cytoplasm or around the nucleus were as positive. The positive cells accounted for 50% of total cells were regarded as APF-positive，and the results were compared before and after inactivating serum complement. The APF of 76 rheumatoid arthritis（RA） patients at activity and remission periods and 61 controls without RA were determined，and the results were analyzed comparatively with the fluorescent label method（one-step method） for anti-human IgG APF. Results　The positive rates of both methods had no significant differences before and after inactivating serum complement（P＞0.05）. The positive rates of the 2 methods were significantly different（P＜0.05）. Two-step method surpassed the one-step method in sensitivity（75.0%），specificity（96.7%） and positive predictive value（96.6%） which were higher than those of the one-step method. The APF fluorescence intensity in activity period was higher than that in remission period in the identical patients with RA. Conclusions　Two-step method can detect APF antibody of IgM，IgA and IgG types simultaneously，and surpasses the one-step method in resolution，sensitivity and specificity. With rheumatoid factor（RF） and anti-keratin antibodies（AKA），they can complement each other in diagnosis of RA and improve the diagnosis of RA.

Key words: Anticomplement C_3C, Fluorescent label, Anti-perinuclear factor, Rheumatoid arthritis