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Generation and Characterization of a Novel Recombinant Antibody Against 15-Ketocholestane Isolated by Phage-Display
Immunology Program, Department of Microbiology, Yong Loo Lin School of Medicine, Centre for Life Sciences, National University of Singapore, 117456 Singapore
Department of Biochemistry, 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
* Author to whom correspondence should be addressed.
Received: 1 March 2012; in revised form: 27 March 2012 / Accepted: 11 April 2012 / Published: 19 April 2012
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.
Keywords: phage display; oxysterols; multiple sclerosis; mass-spectrometry
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Islam, M.O.; Lim, Y.T.; Chan, C.E.Z.; Cazenave-Gassiot, A.; Croxford, J.L.; Wenk, M.R.; Macary, P.A.; Hanson, B.J. Generation and Characterization of a Novel Recombinant Antibody Against 15-Ketocholestane Isolated by Phage-Display. Int. J. Mol. Sci. 2012, 13, 4937-4948.
Islam MO, Lim YT, Chan CEZ, Cazenave-Gassiot A, Croxford JL, Wenk MR, Macary PA, Hanson BJ. Generation and Characterization of a Novel Recombinant Antibody Against 15-Ketocholestane Isolated by Phage-Display. International Journal of Molecular Sciences. 2012; 13(4):4937-4948.
Islam, Md. Omedul; Lim, Yan Ting; Chan, Conrad En Zuo; Cazenave-Gassiot, Amaury; Croxford, J. Ludovic; Wenk, Markus R.; Macary, Paul A.; Hanson, Brendon J. 2012. "Generation and Characterization of a Novel Recombinant Antibody Against 15-Ketocholestane Isolated by Phage-Display." Int. J. Mol. Sci. 13, no. 4: 4937-4948.