OBJECTIVE To generate and characterize the anti-anti-idiotypic monoclonal antibody (MAb) of epithelial ovarian carcinoma (EOC)ìwith the aim of further investigating the mechanism of active immunotherapy of tumor vaccine. METHODS Using anti-idiotypic MAb (6B11)of EOC as immunogen, the anti-anti-idiotypic MAb 3D12 was generated by hybridoma technology. To characterize the 3D12, ELISA, immunohistochemistry, flow cytometry and western blot were used to define its specific reactivity with both 6B11 and the original EOC antigen OC166-9. RESULTS One hybridoma cell secreting 3D12 was obtained. The 3D12 showed the ability of specific binding to 6B11 and OC166-9, and of inhibiting the binding of COC166-9 (the MAb against OC166-9) to OC166-9. A positive staining of serous papillary EOC and the specific binding of SKOV3 cell line with 3D12 supernatant were also demonstrated. Western blot showed 3D12 and COC166-9 could immunoprecipitate the same protein. The 3D12 was IgM, with the affinity Kaff approximately 8.34x10(7) L/mol. CONCLUSION The anti-anti-idiotypic MAb 3D12 is confirmed as Ab1-like Ab3, which indirectly proves that 6B11 bears the internal image of OC166-9.