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Journal of Lipid Research, Vol. 50, 586-594, March 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Methods

A sensitive and specific ELISA detects methionine sulfoxide-containing apolipoprotein A-I in HDL^*

Xiao Suo Wang^*, Baohai Shao, Michael N. Oda, Jay W. Heinecke, Stephen Mahler^** and Roland Stocker^1,*

^* Centre for Vascular Research, School of Medical Sciences (Pathology) and Bosch Institute, The University of Sydney, Sydney, Australia
Department of Medicine, University of Washington, Seattle
Children's Hospital Oakland Research Institute, Oakland, CA 94609
^** Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Australia

^* This research was supported by Psiron Pty Ltd and a University of Sydney Post-graduate Award Scholarship (to X.S.W.), the National Health & Medical Research Council of Australia (Program grant and Fellowship to R.S.), the University of Sydney Medical Foundation (to R.S.), and by grants from the National Institutes of Health (K99HL091055, HL086798, P30DK017047, and PO1HL030086). Mass spectrometry analyses of recombinant apoA-Is were supported by the Mass Spectrometry Core, Diabetes, and Endocrinology Research Center, University of Washington.

Published, JLR Papers in Press, October 2, 2008.

¹ To whom correspondence should be addressed. e-mail: rstocker{at}med.usyd.edu.au

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 MetO⁸⁶ and MetO¹¹² (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.

Supplementary key words protein oxidation • lipid hydroperoxides • dysfunctional HDL • myeloperoxidase • oxidative stress • reactive nitrogen species

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