We induced EAO by immunization using viable syngeneic TGC without using any adjuvant. B cell infiltration and IgG deposit were detected by immunohistochemistry in the testis of EAO. IgG titers in TGC-induced EAO mice were extremely high as detected by enzyme-linked immunosorbent assay (ELISA). Then, we identified TGC-specific AIs by TGC liquid chromatography–tandem mass spectrometry (MS) analysis, followed by two-dimensional gel electrophoresis (2D), which showed serum IgG from EAO mice reaction. The expression pattern of identified TGC-specific AIs was analyzed by the Real time PCR. The recombinant proteins of identified 10 (except unnamed protein) TGC-specific AIs were created by using human embryonic kidney 293 (HEK293) cells and these antigencities were reconfirmed by Western blot using EAO serum reaction.