Immunochemical characterization of purified human oxidized low-density lipoprotein antibodies

The goal of this study was to characterize the isotypes and reactivity of h uman autoantibodies to copper oxidized LDL (oxLDL). Forty-six purified oxLD L antibodies contained immunoglobulins of the three major isotypes, with a predominance of IgG;, subclasses 1 and 3. These IgG isotypes are known to i nteract with FcR gamma I and to activate the complement system and thus are potentially able to activate macrophages and cause foam cell formation. Th e same purified antibodies were tested for cross-reactivity with malondiald ehyde (MDA)-, glycated (Glyc)-, and native (n)LDL and cardiolipin. Absorpti on with oxLDL resulted in a decrease of reactivity of 77.2 +/- 4.7%. Absorp tion with MDA-LDL resulted in a wider range of reduction of reactivity valu es, ranging from 50 to 87%, possibly reflecting differences in the degree o f MDA modification. Absorption with Glyc- and nLDL caused a minor decrease in the reactivity of antibodies to oxLDL (5.9 +/- 7.1 and 6.8 +/- 6.4%, res pectively), comparable to the reduction of reactivity (2.1 +/- 4.0%) measur ed after absorption with transferrin, an irrelevant protein used as a negat ive control. These results suggest that oxLDL antibodies recognize primaril y MDA epitopes. To determine whether purified oxLDL antibodies also recogni ze other epitopes known to be generated during copper oxidation of LDL, suc h as 4-hydroxynonenal (HNE- and N''(carboxymethyl)-lysine (CML), two additi onal sets of experiments were carried out. First, we monitored the formatio n of CML-, MDA-lysine, and HNE-lysine at different times during copper oxid ation of two LDL pools. Both pools showed simultaneous increases in protein modification, as indicated by increasing fluorescence emission at 430 nm, and in immunoreactivity with oxLDL antibodies, coinciding closely with MDA modification of lysine groups. Second, we assessed whether the reactivity o f oxLDL antibodies could be blocked by absorption with CML- or HNE-LDL. HNE -LDL did not react with isolated oxLDL antibodies. Highly modified CML-LDL (>90% of lysine residues modified) reduced the reactivity of oxLDL antibodi es, but only by 25.5%. Finally, we investigated the possible cross-reactivi ty of oxLDL antibodies with cardiolipin. Seventeen purified oxLDL antibodie s were used in this study, which showed that absorption with oxLDL or nLDL did not affect their reactivity with immobilized cardiolipin, (C) 2000 Acad emic Press.