Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL.
Lipid hydroperoxide may react with protein or amino phospholipid without secondary decomposition. We prepared a polyclonal antibody to lipid hydroperoxide-modified proteins using 13S-hydroperoxy-9Z, 11E-octadecadienoic acid-modified keyhole limpet hemocyanin (13-HPODE-KLH) as immunogen. The antibody recognized 13-HPODE-modified bovine serum albumin (BSA), but not aldehyde-modified proteins, such as malondialdehyde-modified BSA. The antibody also recognized adducts derived from 13-HPODE and 13S-hydroperoxy-9Z, 11E, 15Z-octadecatrienoic acid (13-HPOTRE(alpha)). The oxidized alpha-linolenic acid- and 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 observed 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. The results suggest that lipid hydroperoxide-modified apolipoprotein was formed during the oxidation of LDL.
Kato
Y
Y
Department of Applied Biological Sciences, Nagoya University, Japan.
Makino
Y
Y
Osawa
T
T
D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D000941:Antigens / Q000737:chemistry / Q000276:immunology; D053299:Apolipoprotein B-100; D001053:Apolipoproteins / Q000737:chemistry; D001055:Apolipoproteins B / Q000737:chemistry / Q000276:immunology*; D006433:Hemocyanins / Q000276:immunology; D015151:Immunoblotting; D008041:Linoleic Acids / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D008054:Lipid Peroxides / Q000737:chemistry* / Q000276:immunology*; D008077:Lipoproteins, LDL / Q000737:chemistry / Q000276:immunology*; D008670:Metals / Q000494:pharmacology; D010446:Peptide Fragments / Q000276:immunology; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000276:immunology
D000906:Antibodies; D000941:Antigens; D053299:Apolipoprotein B-100; D001053:Apolipoproteins; D001055:Apolipoproteins B; D008041:Linoleic Acids; D008054:Lipid Peroxides; D008077:Lipoproteins, LDL; D008670:Metals; D010446:Peptide Fragments; C099252:oxidized low density lipoprotein; C038649:13-hydroperoxy-9,11-octadecadienoic acid; D012710:Serum Albumin, Bovine; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin