title: A sensitive and specific ELISA detects methionine sulfoxide-containing apolipoprotein A-I in HDL.
abstract: Oxidized HDL has been proposed to play a key role in atherogenesis. A wide range of reactive intermediates oxidizes methionine residues to methionine sulfoxide (MetO) in apolipoprotein A-I (apoA-I), the major HDL protein. These reactive species include those produced by myeloperoxidase, an enzyme implicated in atherogenesis. The aim of the present study was to develop a sensitive and specific ELISA for detecting MetO residues in HDL. We therefore immunized mice with HPLC-purified human apoA-I containing MetO(86) and MetO(112) (termed apoA-I(+32)) to generate a monoclonal antibody termed MOA-I. An ELISA using MOA-I detected lipid-free apoA-I(+32), apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Detection was concentration dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radical or metal ions, or LDL and methionine-containing proteins other than apoA-I modified by 2e-oxidants. Because the ELISA we have developed specifically detects apoA-I containing MetO in HDL and plasma, it should provide a useful tool for investigating the relationship between oxidized HDL and coronary artery disease.
authors.affiliation: Centre for Vascular Research, School of Medical Sciences (Pathology) and Bosch Institute, The University of Sydney, Sydney, Australia.
authors.forename: Xiao Suo
authors.initials: XS
authors.lastname: Wang
authors.affiliation: Department of Medicine, University of Washington, Seattle.
authors.forename: Baohai
authors.initials: B
authors.lastname: Shao
authors.affiliation: Children's Hospital Oakland Research Institute, Oakland, CA 94609.
authors.forename: Michael N
authors.initials: MN
authors.lastname: Oda
authors.affiliation: Department of Medicine, University of Washington, Seattle.
authors.forename: Jay W
authors.initials: JW
authors.lastname: Heinecke
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Australia.
authors.forename: Stephen
authors.initials: S
authors.lastname: Mahler
authors.affiliation: Centre for Vascular Research, School of Medical Sciences (Pathology) and Bosch Institute, The University of Sydney, Sydney, Australia. Electronic address: rstocker@med.usyd.edu.au.
authors.forename: Roland
authors.initials: R
authors.lastname: Stocker
mesh_terms: D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I / Q000097:blood / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D001774:Blood Chemical Analysis / Q000379:methods / Q000706:statistics & numerical data; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D008075:Lipoproteins, HDL / Q000097:blood / Q000737:chemistry*; D008297:Male; D008715:Methionine / Q000031:analogs & derivatives* / Q000032:analysis; D051379:Mice; D008875:Middle Aged; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D011994:Recombinant Proteins; D008715:Methionine; C013111:methionine sulfoxide