Immunologic detection and measurement of hypochlorite-modified LDL with specific monoclonal antibodies.
Oxidation of LDL is thought to contribute to the early stages of atherogenesis. Because myeloperoxidase is present in atherosclerotic lesions and can produce the strong oxidant hypochlorous acid (HOCl), which converts LDL into its high-uptake atherogenic form in vitro, we raised polyclonal and monoclonal antibodies (MoAbs) against HOCl-modified LDL (HOCl-LDL). Characterization of the polyclonal anti-human HOCl-LDL Abs showed that they cross-reacted strongly with 4-hydroxynonenal-, malondialdehyde-, and Cu(2+)-oxidized LDL. Similarly, polyclonal and some monoclonal Abs against aldehyde- and Cu(2+)-modified LDL cross-reacted with HOCl-LDL. In contrast to the polyclonal Abs, two selected hybridoma cell line supernatants containing MoAbs raised against HOCl-LDL (MoAb-A and MoAb-B) did not cross-react with either native LDL or aldehyde- or Cu(2+)-modified LDL. MoAb-A (clone 1B10A11, subtype IgG1 kappa) recognized an epitope that appeared to be specific for HOCl-LDL and depended on the tertiary structure of the (lipo)protein, as judged by a lack of cross-reactivity with HOCl-modified human and bovine serum albumin and a loss of reactivity associated with lipoprotein denaturation. MoAb-B (clone 2D10G9, subtype IgG2b kappa), on the other hand, gave identical titration curves with HOCl-LDL and HOCl-modified albumins, suggesting that this antibody recognized epitopes that are commonly generated on proteins that have been oxidized with HOCl. Thus, MoAb-A and MoAb-B may be useful tools for the investigation of a possible role for HOCl-mediated damage to (lipo)proteins in atherosclerosis and other inflammatory diseases.
Malle
E
E
Karl-Franzens University, Institutes of Medical Biochemistry, Graz, Austria.
Hazell
L
L
Stocker
R
R
Sattler
W
W
Esterbauer
H
H
Waeg
G
G
D000447:Aldehydes / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001667:Binding, Competitive; D015153:Blotting, Western; D003300:Copper / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006997:Hypochlorous Acid / Q000494:pharmacology*; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000032:analysis* / Q000737:chemistry; D011817:Rabbits; D012709:Serum Albumin / Q000276:immunology
D000447:Aldehydes; D000911:Antibodies, Monoclonal; D000939:Epitopes; D008077:Lipoproteins, LDL; D012709:Serum Albumin; D006997:Hypochlorous Acid; D003300:Copper