Localization of isoketal adducts in vivo using a single-chain antibody.
Isoketals are highly reactive gamma-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-E2-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-F2t-isoprostane (8-iso-prostaglandin F2alpha). 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.
Davies
Sean S
SS
Department of Pharmacology, Vanderbilt University, Nashville, TN 37232-6602, USA. sean.davies@vanderbilt.edu
Talati
Megha
M
Wang
Xiahong
X
Mernaugh
Raymond L
RL
Amarnath
Venkataraman
V
Fessel
Joshua
J
Meyrick
Barbara O
BO
Sheller
James
J
Roberts
L Jackson
LJ
D000447:Aldehydes / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D000818:Animals; D000918:Antibody Specificity; D004847:Epithelial Cells / Q000276:immunology; D018604:Epitope Mapping; D005260:Female; D018496:Hyperoxia / Q000378:metabolism; D007120:Immunochemistry; D007135:Immunoglobulin Variable Region / Q000276:immunology* / Q000302:isolation & purification; D015227:Lipid Peroxidation; D008168:Lung / Q000166:cytology / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D015394:Molecular Structure; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D011453:Prostaglandins / Q000737:chemistry* / Q000378:metabolism; D011458:Prostaglandins E / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D011506:Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification
D000447:Aldehydes; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D010455:Peptides; D011453:Prostaglandins; D011458:Prostaglandins E; D011506:Proteins; D011994:Recombinant Proteins; C047684:levuglandin E2