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Mouse monoclonal antibodies against analogues of two mycotoxins (deoxynivalenol (DON) and nivalenol (NIV)) commonly found in cereal crops were isolated and used to develop an immunological detection assay. We immunized BALB/c mice with 4,15-diacetylnivalenol-3-O-hemisuccinate conjugated to keyhole limpet hemocyanin and isolated 21 hybridomas secreting monoclonal antibody (MAB) which reacted with 3,4,15-triacetylnivalenol (3,4,15-TANIV). These MABs were classified into two distinct types of reactivity against analogues of NIV and DON. Two monoclonal antibodies, KTM-205 and 208, were highly specific for 3,4,15-TANIV, whereas KTM-233, 239 and 240, cross-reacted with both 3,15-diacetyl deoxynivalenol (3,15-DADON) and 3,4,15-TANIV to the same extent. Acetylation under mild conditions can derivatize nivalenol and deoxynivalenol to 3,4,15-TANIV and 3,15-DADON, respectively. Thus the competitive indirect ELISAs for measuring nivalenol and deoxynivalenol were developed using these monoclonal antibodies. The detectable level of 3,4,15-triacetylnivalenol and 3,15-diacetyldeoxynivalenol was about 0.3 to 1000 pg/mL in buffer by the indirect competitive ELISA with KTM-240. Thus we have successfully generated useful MABs for an ELISA for the detection of nivalenol and deoxynivalenol in agricultural crops.