cholest-8(14)-en-3-ol-15-one and Multiple-Sclerosis

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.

Topics: Animals; Antibodies, Monoclonal; Biomarkers; Cell Surface Display Techniques; Cholestenones; Cholesterol; Encephalomyelitis, Autoimmune, Experimental; Humans; Mice; Multiple Sclerosis; Peptide Library

15-Oxygenated cholesterol species such as 5α-cholest-8(14)ene-3β,15α-diol (15HC) and 3β-hydroxy-5α-cholest-8(14)-en-15-one (15KC) are commercially available synthetic products unlikely to occur in biological systems. Surprisingly, Farez et al. recently reported that these two steroids occur in human circulation at levels considerably higher than those of any other endogenous oxysterol [Farez, M. et al. 2009. Toll-like receptor 2 and poly(ADP-ribose) polymerase 1 promote central nervous system neuroinflammation in progressive EAE. Nat. Immunol. 10: 958-964]. The levels were reported to be increased in patients with multiple sclerosis in a progressive phase and the authors suggested that this could be utilized diagnostically. Based on extensive in vitro experiments exposing cells to the same high levels of 15HC as found in vivo (1000 ng/ml) the authors concluded that 15HC may be an important pathogenetic factor in multiple sclerosis. Using combined gas chromatography-mass spectrometry we fail to detect significant plasma levels of 15HC either in healthy controls or in patients with multiple sclerosis (levels < 2 ng/ml). If 15KC is present in these plasma samples, the concentration of it must be <10 ng/ml. Our failure to detect significant levels of the above steroids could not be due to loss during hydrolysis and work-up because recovery of the added two oxysterols was close to 100%. Autoxidation of lipoprotein-bound cholesterol resulted in extensive conversion of cholesterol into 7-oxygenated but not 15-oxygenated sterols. We conclude that if present there are trace amounts only of the above 15-oxygenated steroids in human circulation and that the role of such oxysterols as pathogenetic factors and biomarkers must be reconsidered.

Topics: Adult; Cholestenones; Female; Humans; Hydroxycholesterols; Male; Middle Aged; Multiple Sclerosis; Oxidation-Reduction; Oxygen