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Wang XS, Shao B, Oda MN, Heinecke JW, Mahler S, Stocker R: A sensitive and specific ELISA detects methionine sulfoxide-containing apolipoprotein A-I in HDL. J Lipid Res. 2009 Mar;50(3):586-94. Epub 2008 Oct 2.
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. |
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