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 protein oxidation. We prepared an antibody specific for protein dityrosine 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 H2O2/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. (C) 2000 Academic Press.