title: Generation and characterization of a novel recombinant antibody against 15-ketocholestane isolated by phage-display.
abstract: The employment of monoclonal antibodies (Mabs) to identify disease-associated biomarkers in clinical samples represents the underlying principle for many diagnostic tests. To date, these have been principally developed for protein targets with few reported applications for lipids due to their hydrophobicity and poor immunogenicity. Oxysterols represent a family of lipids implicated in diverse human diseases where Mab-based detection assays could have a profound effect on their utility as clinical biomarkers. These are usually identified in patients' samples by mass- spectrometry based approaches. Here, we describe an antibody phage-library based screening methodology for generating a recombinant monoclonal antibody (RAb) targeting the oxysterol-15-ketocholestane (15-KA), a lipid implicated in multiple sclerosis and Autoimmune Encephalomyelitis (EAE). The antibody is highly specific for 15-KA and shows little or no binding activity for other closely related oxysterols. We employ RAb2E9 to address the controversy over whether 15-KA is a true biomarker for MS/EAE and show that 15-KA is undetectable in serum taken from mice with EAE using antibody based detection methodologies; a finding confirmed by mass-spectrometry analysis. This study demonstrates the technical feasibility of using phage display to isolate highly specific antibodies against poorly immunogenic, small molecule lipids.
authors.affiliation: Immunology Program, Department of Microbiology, Yong Loo Lin School of Medicine, Centre for Life Sciences, National University of Singapore, 117456 Singapore.
authors.forename: Md Omedul
authors.initials: MO
authors.lastname: Islam
authors.affiliation: Immunology Program, Department of Microbiology, Yong Loo Lin School of Medicine, Centre for Life Sciences, National University of Singapore, 117456 Singapore.
authors.forename: Yan Ting
authors.initials: YT
authors.lastname: Lim
authors.affiliation: Immunology Program, Department of Microbiology, Yong Loo Lin School of Medicine, Centre for Life Sciences, National University of Singapore, 117456 Singapore.|Defence Medical and Environmental Research Institute, DSO National Laboratories, 117510 Singapore, Singapore.
authors.forename: Conrad En Zuo
authors.initials: CEZ
authors.lastname: Chan
authors.affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, Centre for Life Sciences, National University of Singapore, 117456 Singapore.
authors.forename: Amaury
authors.initials: A
authors.lastname: Cazenave-Gassiot
authors.affiliation: Immunology Program, Department of Microbiology, Yong Loo Lin School of Medicine, Centre for Life Sciences, National University of Singapore, 117456 Singapore.
authors.forename: J Ludovic
authors.initials: JL
authors.lastname: Croxford
authors.affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, Centre for Life Sciences, National University of Singapore, 117456 Singapore.
authors.forename: Markus R
authors.initials: MR
authors.lastname: Wenk
authors.affiliation: Immunology Program, Department of Microbiology, Yong Loo Lin School of Medicine, Centre for Life Sciences, National University of Singapore, 117456 Singapore.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Macary
authors.affiliation: Immunology Program, Department of Microbiology, Yong Loo Lin School of Medicine, Centre for Life Sciences, National University of Singapore, 117456 Singapore.|Defence Medical and Environmental Research Institute, DSO National Laboratories, 117510 Singapore, Singapore.
authors.forename: Brendon J
authors.initials: BJ
authors.lastname: Hanson
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D015415:Biomarkers / Q000097:blood*; D061505:Cell Surface Display Techniques; D002783:Cholestenones / Q000097:blood / Q000276:immunology*; D002784:Cholesterol / Q000031:analogs & derivatives / Q000276:immunology; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000175:diagnosis*; D006801:Humans; D051379:Mice; D009103:Multiple Sclerosis / Q000175:diagnosis*; D019151:Peptide Library
chemical_list: D000911:Antibodies, Monoclonal; D015415:Biomarkers; D002783:Cholestenones; D019151:Peptide Library; C015073:cholest-8(14)-en-3-ol-15-one; D002784:Cholesterol