Generation of a polyclonal antibody against lipid peroxide-modified proteins

A specific polyclonal antibody against the lipid peroxide (LOOH)-modified rabbit serum albumin (RSA) was generated in rabbits. The antibody selectively recognized the modified protein in a concentration-dependent manner and did not cross react with aldehyde-modified proteins or proteins directly oxidized with the free radical generator 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH). Oxidized low-density lipoprotein (Ox-LDL), but not native LDL, was also recognized by the antibody in a concentration-dependent manner. The antibody also cross reacted with several other proteins modified by LOOH suggesting that the antibody is directed towards a common epitope and not towards the protein sequence. Western blot analysis of normal human plasma showed that at least three different proteins are recognized by the antibody. RAW cells, preincubated with LOOH, were immunostained with the antibody and the antigenic epitopes were present intracellularly, while controls lacking in the primary antibodies failed to show immunoreactivity. Atherosclerotic arteries from cholesterol-fed monkeys and human atherosclerotic lesions were also immunostained by the antibody. The immunoreactivity was co-localized in areas rich in foam cell macrophages. These results suggest that LOOH-modified proteins present an unique antigenic epitope that may represent a primary product of interaction of LOOH with proteins.