An antibody for 4-nonylphenol (NP), which is suspected to be an endocrine disrupting compound, was perpared. First, an amino group was introduced at the ortho-position of NP (mixture of branched alkyl chain compounds), and the NP-protein conjugates were prepared. When NP was measured using antibody raised with the conjugate as an immunogen by an indirect competitive ELISA, the antibody showed an affinity with NP, but the magnitude of the affinity was weak. The antibody showed stronger affinity with NP having branched alkylgroup than with NP having straight chain. Next, an attention was paid to the position of hydroxyl- and alkyl-group on NP, and new immunogens were prepared. The antibodies raised with newly prepared immunogens, however, showed only very weak affinity with NP. These results sggested that the antibody have stronger affinity with the solid phase antigen than with free NP, resulting weak competition. In order to elucidate the recognition property of the antibody against the antigen, the cross-reactivity of the antibody against various kinds of antigens were investigated. As the results, the antibody did recognize an oxygen atom or a hydroxyl-group on the benzene ring. Based on these results, a solid phase antigen (NP-O-hexanoate-ovalbumin conjugate) having the structure including oxygen atom on the benzene ring was prepared. By the measuring system using NP-O-hexanoate-ovalbumin conjugate as a solid phase antigen, the antibody showed affinity with free NP. For sensitive measurement, the combination of a solid phase antigen, free antigen and an antibody is the most important factors in an indirect competitive immunoassay.