Model IgG Monoclonal Autoantibody–Anti-Idiotype Pair for Dissecting the Humoral Immune Response to Oxidized Low Density Lipoprotein

Abstract

Increasing evidence implicates IgG autoantibodies against oxidized forms of low density lipoprotein (oxLDL) in the pathophysiology of atherosclerotic arterial disease. However, insufficient knowledge of their structure and function is a key gap. Using an elderly LDL receptor-deficient atherosclerotic mouse, we isolated a novel IgG3k against oxLDL (designated MAb LO1). LO1 reacts with copper-oxidized LDL, but minimally with native LDL. Further analysis showed that MAb LO1 also reacts in vitro with malondialdehyde-conjugated LDL (MDA-LDL), a known key epitope in copper-oxidized LDL preparations. By screening a phage library expressing single chain variable region antibodies (scFv), we selected an anti-idiotype scFv (designated H3) that neutralizes MAb LO1 binding to MDA-LDL. Amino acid substitutions between H3 and an irrelevant control scFv C12 showed that residues in the H3 CDRH2, CDRH3, and CDRL2 are all critical for MAb LO1 binding, consistent with a conformational epitope on H3 involving both heavy and light chains. Comparison of amino acids in H3 CDRH2 and CDRL2 with apoB, the major LDL protein, showed homologous sequences, suggesting H3 has structural similarities to the MAb LO1 binding site on MDA-LDL. Immunocytochemical staining showed that MAb LO1 binds epitopes in mouse and human atherosclerotic lesions. The MAb LO1-H3 combination therefore provides a very promising model for analyzing the structure and function of an individual IgG autoantibody in relation to atherosclerosis.

Near Infrared Fluorescence (NIRF) Molecular Imaging of Oxidized LDL with an Autoantibody in Experimental Atherosclerosis

“…LO1-750 staining of human carotid atherosclerotic plaque showed similar specific diffuse and focal staining ( Fig. 2C-H ) to that previously shown with the unlabeled antibody 21 . As was the case in our previous study with unconjugated LO1 21 , binding of LO1-750 to atherosclerotic lesions was inhibited by MDA-LDL in solution (data not shown).…”

“…Staining with control IgG3 was negative. Overall, the localization of the unlabeled antibody to coronary lesions was in a pattern similar to that we have previously shown in human carotid atherosclerosis 21 .…”

“…2C-H ) to that previously shown with the unlabeled antibody 21 . As was the case in our previous study with unconjugated LO1 21 , binding of LO1-750 to atherosclerotic lesions was inhibited by MDA-LDL in solution (data not shown).…”

“…The LO1 antibody that we used reacts with LDL adducted with MDA, and was originally isolated from an Ldlr −/− mouse that had not been subjected to active immunization, giving us the confidence that it is likely to target a biologically relevant endogenous entity. Furthermore, the genes encoding the LO1 heavy and light chain are essentially germline, indicating that it is a naturally occurring autoantibody 21 .…”

“…LO1 is a spontaneously arising IgG3k germline encoded natural monoclonal autoantibody isolated in our laboratory. It was selected for reacting with copper-oxidized LDL in vitro, and subsequently found to recognize LDL conjugated with malondialdehyde (MDA-LDL) 21 . LO1 recognizes antigen in tissue sections of mouse and human atherosclerosis upon immunocytochemical staining, with the binding to sections prevented by free MDA-LDL 21 .…”