〔Abstract〕 Objective To investigate the antigenic change and its clinical significance of the cyclized inner lipoyl domain(ILD) in the E2 subunit of pyruvate dehydrogenase complex(PDC-E2) for detection of anti-mitochondrial antibodies(AMA) in patients with primary biliary cholangitis(PBC). Methods Cyclized ILD(cILD) was obtained through its construction, expression and purification by intein self-cleavage process provided by plasmid pTWIN1. Western blot was employed to identify the antigenicity of the recombinant linear ILD and cILD. A total of 316 patients with PBC, 34 patients with rheumatoid arthritis(RA) and 36 healthy subjects(HC) were involved in this study. The AMA in their serum was comparatively detected by enzyme linked immunosorbent assay(ELISA), in which PDC-E2, linear ILD and cILD were respectively used as coating antigens. Results The immunogenicity to AMA of both the recombinant linear ILD and cILD was demonstrated by Western blot. The positive rates of AMA detected by ELISA with PDC-E2, linear ILD and cILD as coating antigens were 93.04%(294/316), 91.46%(289/316) and 85.76%(271/316), respectively. The positive rate of AMA detected by cILD as coating antigen was significantly lower than that detected by PDC-E2 and linear ILD as coating antigens(85.76%vs93.04%,P=0.003; 85.76%vs93.04%,P=0.024). There was no significant difference in AMA positive rate for the PDC-E2 and linear ILD as coating antigens(93.04%vs91.46%,P=0.744). In 21 PBC patients with negative AMA to both PEC-E2 and linear ILD, the AMA positive rate was 38.10%(8/21) when cILD was taken as coating antigen. Conclusion Although with some decrease in cILD antigenicity to AMA, cILD may have potential new clinical value in the diagnosis of PBC with negative AMA.