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Immunochemical detection of protein dityrosine in atherosclerotic lesion of apo-E-deficient mice using a novel monoclonal antibody.

Dityrosine is one of the specific biomarkers for proteinoxidation. We prepared an antibody specific for proteindityrosine using a dimer of 3-p-(hydroxyphenyl)propionic acid (di-HP) as a hapten. Three clones (A8, G6, and 1C3) were obtained, and the antibody from the A8 clone reacted with the di-HP-conjugated protein but not with a dityrosine conjugate. The others (G6 and 1C3 clones) recognized both the di-HP and dityrosine conjugates. The antibodies reacted with peptidyl dityrosine, derived from Thr-Tyr-Ser, rather than the free dityrosine. The reactivity of the latter two antibodies with lens proteins oxidized by incubation with H(2)O(2)/Cu was in good accordance with the formation of the dityrosine-like fluorescence. Using the obtained monoclonal antibody, the immunopositive staining of atherosclerotic lesions in Apo E-deficient mice was confirmed by an immunohistochemical technique.