A sensitive and specific ELISA detects methionine sulfoxide-containing apolipoprotein A-I in HDL.
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.
Wang
Xiao Suo
XS
Centre for Vascular Research, School of Medical Sciences (Pathology) and Bosch Institute, The University of Sydney, Sydney, Australia.
Shao
Baohai
B
Department of Medicine, University of Washington, Seattle.
Oda
Michael N
MN
Children's Hospital Oakland Research Institute, Oakland, CA 94609.
Heinecke
Jay W
JW
Department of Medicine, University of Washington, Seattle.
Mahler
Stephen
S
Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Australia.
Stocker
Roland
R
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.
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
D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D011994:Recombinant Proteins; D008715:Methionine; C013111:methionine sulfoxide