Abstract
Isoketals are highly reactive γ-ketoaldehydes formed by the oxidation of arachidonic acid that rapidly adduct to proteins. To investigate the formation of isoketal adducts in vivo, we isolated and characterized a single-chain antibody from a phage displayed recombinant ScFv library that bound a model peptide adducted with synthetic 15-E 2-isoketal. Recognition of isoketal adduct by this anti-isoketal adduct single-chain antibody was essentially independent of the amino acid sequence of adducted peptides or proteins. The antibody did not cross-react with 4-hydroxynonenal or 4-oxononanal adducts or with 15-F 2t-isoprostane (8-iso-prostaglandin F 2α). We investigated the formation of isoketal adducts in a well-established model of oxidative injury, hyperoxia. Exposure to >98% oxygen for 7 h dramatically increased both the number of immunoreactive airway epithelial cells and the intensity of immunoreactivity compared with animals exposed to normal room air (21% oxygen). We conclude that isoketal adducts form in epithelial cells as a result of high oxygen exposure and that this single-chain antibody provides a valuable tool to localize the formation of isoketal adducts in tissues in vivo.
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