Presence of phospholipid-neutral lipid complex structures in atherosclerotic lesions as detected by a novel monoclonal antibody

A novel monoclonal antibody (ASH1a/256C) that recognizes atherosclerotic le sions in human and Watanabe heritable hyperlipidemic (WHHL) rabbit aortae i s described. When I-123-labeled ASH1a/256C antibody is injected intravenous ly into WHHL rabbits, it associates specifically with fatty streaks on the aorta. The antigen recognized by the antibody is lipid, based on extraction with chloroform and methanol from WHHL rabbit tissues. The antigen, purifi ed by high performance liquid chromatography, was shown to be phosphatidylc holine (PC), which contains unsaturated fatty acyl groups based on analyses utilizing H-1 and C-13 nuclear magnetic resonance, Fourier transfer-infrar ed spectrum, and mass spectrometry, The antibody did not react with other c lasses of phospholipids or neutral lipids when tested using an enzyme-linke d immunosorbent assay. When PC was mixed with either cholesterol, cholester yl ester, or triacylglycerol, however, the reactivity of the antibody to PC increased up to 8-fold. Homogenates of aorta tissue obtained from normal a nd WHHL rabbits were fractionated using sucrose density gradient ultracentr ifugation in which neutral lipid droplets, cellular membranes, and proteins are separated. The phospholipid content in cellular membrane fractions fro m WHHL rabbits was twice as high as that of normal rabbits, and there was a n enormous difference in the antigenic activity in these fractions. The con tent of cholesterol in the cellular membrane fraction of WHHL rabbits was a pproximately 50 times higher than that of normal rabbits. Addition of neutr al lipids to the cellular membrane fraction of normal rabbit markedly incre ased the antigenic activity. Atheromatous lesions in thickened WHHL rabbit aortic intima that were rich in lipid droplets were stained positively with ASH1a/256C immunohistochemically. These results strongly suggest that PC-n eutral lipid complex domains are formed in atherosclerotic lesions.