Immunoassays using specific antibodies, including those targeting ZEN, have been used to detect toxin residues for many years.The key step in the development of an immunoassay is the production of the specific antibody.But the cross-reactivity (CR) of anti-ZEN antibodies with α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL) is a serious problem.ZEN and its 5 analogs are structurally identical apart from minor differences at the C7 and C12 positions in the macrocyclic lactone ring.Therefore it is difficult for an anti-ZEN antibody to distinguish ZEN from its 5 analogs.Anti-ZEN anti-bodies showed high cross-reactivity (CR) with the 5 analogsTo overcome this problem and improve the specificity of immunoassays, we produced anti-ZEN antibodies based on a ZEN-cationic protein conjugate.In this study, ZEN was coupled with cationic bovine serum albumin (cBSA) via a Mannich reaction.After BALB/c mice were immunized with ZEN-cBSA, an immunological response was rapidly induced.The titers of the polyclonal antisera and monoclonal antibody were 30,000 and 20,000, respectively.Cross-reactivity (CR) values of the anti-ZEN polyclonal antisera and monoclonal antibody with the 5 analogs were <7% and <2%, respectively.An indirect competitive enzyme-linked immunosorbent assay based on the monoclonal anti-ZEN antibody was established.The recovery rates of ZEN in spiked cereal and feed were in the range of 80%-120% with coefficients of variation <15%.The intra-assay variation and inter-assay variation in assay buffer were both <5%.Therefore, the results demonstrated a feasible approach for preparing highly specific, higher titer and more rapidly induced antibodies against ZEN by using a ZEN-cBSA conjugate as the immunogen instead of currently used immunogens.