Characterization of a specific polyclonal antibody

Lipid hydroperoxide may react with protein or amino phospholipid without secondary decomposition. We prepared a polyclonal an tibody to lipid hydroperoxide-modified proteins using 13S-hydroperoxy-9Z, 1 1E-octadecadienoic acid-modified keyhole limpet hemocyanin (13-HPODEKLH) as immunogen. The antibody recognized 13-HPODEmodified bovine serum albumin (BSA), but not aldehydemodified proteins, such as malondialdehyde-modified BSA. The antibody also recognized adducts derived from 13HPODE and 13shydroperoxy-9Z, 1 lE, 15Z-octadecatrienoic acid (13-HPOTRE(a)). The oxidized a-linolenic acidand linoleate-protein adducts were recognized by the antibody. Oxidized phospholipid-protein adducts were scarcely recognized by the antibody. However, when ester bonds of phospholipids containing linoleic acid were hydrolyzed by alkaline treatment, the cross-reactivities appeared. The result suggests that a phospholipid hydroperoxide can react with a protein directly or indirectly, and a carboxyl terminal (COOH) of the lipid in an adduct was needed as an epitope. Oxidized LDL (ox-LDL) was prepared by the incubation of LDL with copper ion or 2,2’-azobis(2-amidinopropane)dihydrochloride (AAPH), and the formation of lipid hydroperoxide-modified apolipoprotein was confirmed using the antibody. A slight immunoreactivity was ohsewed in ox-LDL, without alkaline treatment. When the ox-LDL was treated with alkali to hydrolyze the ester bonds of the lipid, enhanced antigenicity appeared with time-dependency.l The results suggest that lipid hydroperoxide-modified apolipoprotein was formed during the oxidation of LDL.-Kato, Y., Y. Makino, and T. Osawa. Characterization of a specific polyclonal antibody against 13-hydr-operoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB100 in oxidized LDI.. ,I. Lipid Hrs. 1997. 38: 1334-1346. Supplementary keywords oxidr protein modification antibody oxidi7ed LDI, lipid hydroperLipid peroxidation probably contributes to the initiation, promotion, and/or progression of some diseases, such as atherosclerosis. The oxidation of the lipid causes the formation of secondary decomposition products including malondialdehyde (MDA) and 4-hydroxy2-nonenal (HNE) . These aldehydes covalently react with protein or nucleic acid (1). The antibodies to MDAor HNEmodified proteins have already been prepared by some workers and used for an evaluation of the oxidative modification of proteins in vivo and in vitro (2-8). The mechanism of the reaction between lipid hydroperoxide and amine is unknown compared to that of the aldehyde-derived modification. Shimasaki, Ueta, and Privett (9) examined the modification of protein by linoleic acid hydroperoxide. The formation of lipofuscin-like fluorescent adducts has been shown as crosslinked polymers. Fruebis, Parthasarathy, and Steinberg (10) suggest that the linoleic acid hydroperoxide reacts with the polypeptide or phosphatidylethanolamine, followed by the formation of a fluorescent compound without the formation of low molecular weight aldehydes, such as HNE or MDA. Hidalgo and Zamora Abbre\.iations: MDA, malondialdehyde; HNE, 4-hydroxy-2-nonenal; LDL, low density lipoprotein; ox-LDL, oxidized LDL; KLH, kcyhole limpet hemocyanin; 9-HPODE, YS-hydroperoxy-lOE, 12Z-octaderadicnoic acid; 13-HPOTRE (a), 1 YS-hydroperoxy-92, 1 IE, 1.52octadecatrienoir acid; 13-HPOTRE (y), 13s-hydroperoxy-62, 9%, 1 1 E-octadecatrienoic acid; 13-HPODE, 1%-hydroperoxy-92, 1 1 E-octadecadienoic acid; AAF’H, 2,2’-aLobis (2-amidinopropane) dihydrochloride; SLO, soybean lipoxygenase; TLC, thin-layer chromatography; PBS, phosphate-buffered saline; TBARS, thiobarbituric acid-reactive substance; TBA, thiobarbituric acid; TCA, trichloroacetic acid; RSA, bovine serum albumin; 1.5-HPETE, 15s-hydroperoxy5Z, 82, 11%, 13E-eicosatetraenoic acid; ox-BSA, oxidized BSA; ELISA, c~7yine-linked immunosorbent assay; TPBS, PBS containing 0.25% Tween ‘LO; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophuresis; PVDF, polynnylidene difluoride; TTBS, Tris-buffiered saline containing 0.25% Tween20; AGE, advanced glycatioii end products; PC, phosphatidylcholine; DTPA, diethylenetriarninepentaacetic acid; DTT, dithiothreitol. ‘To whom correspondencr should be addrcssed. 1334 Journal of Lipid Research Volume 38, 1997 by gest, on N ovem er 6, 2017 w w w .j.org D ow nladed fom (1 1) reported the production of long-chain pyrrole fatty acid esters form carbonyl-amine reactions in methyl 9,10(Z)-epoxy-13-oxo-11 (E)-octadecenoate or 12,13(Z) -epoxy-9-oxo(E) -octadecenoate/butylamine systems. The preparation of the antibody to oxidized low density lipoprotein (ox-LDL) has also been done. Using the antibodies including anti-aldehyde-modified protein antibodies, the presence of oxidatively modified LDL in atherosclerotic lesions has been reported (2-5). Magi1 et al. (12) prepared monoclonal antibodies that recognize the fatty acid product-modified proteins. They prepared immunogens by the reaction of oxidized linoleate or arachidonate with LDL. However, the precise epitopes are not clear. Itabe et al. ( 1 3) established a monoclonal antibody that recognizes ox-LDL, using the homogenates of the human atherosclerotic plaques of the aortae as the immunogens. The epitope of the antibody is thought to be oxidized phosphatidylcholine and its complex with peptides. We assume that a lipid hydroperoxide reacts with a protein without a serious fragmentation of the fattyacid chain. A novel polyclonal an tibody to lipid hydroperoxide-modified protein was prepared using 13S-hydroperoxy-SZ, 1 1E-octadecadienoic acid (13-HPODE)-modified keyhole limpet hemocyanin (KLH) as the immunogen. The epitopes of this antibody were characterized in detail. EXPERIMENTAL PROCEDURES