Frontiers in Cellular Neuroscience (Mar 2015)
Enrichment of an in vivo phage display repertoire by subtraction for easy identification of pathology biomarkers <br /> <br />
Abstract
Titre de la communication : Enrichment of an in vivo phage display repertoire by subtraction for the easy identification of biomarkers of pathology. Vargas-Sanchez J.K., Vekris A., Mordelet E., Dubourdieu-Cassagno N., Ottones F., Petry K.G and Boiziau C. INSERM U1049, Bordeaux University, Neuroinflammation, Imaging and Therapy of Multiple Sclerosis, 146 rue Leo Saignat, 33 076 Bordeaux cedex, France. Abstract Aim. To develop a method that enriches, after in vivo phage display selection, the repertoire with clones specific for a pathological state. Materials and Methods. In a neuroinflammation model, we performed a subtraction based on DNA hybridization and restriction enzyme digestion of the common clones recovered from the central nervous system (CNS) of healthy and experimental autoimmune encephalomyelitis (EAE) rats. The subtraction efficiency was monitored by massive sequencing and clones were examined for binding specificity onto EAE CNS rat tissues and human blood brain barrier cells (hCMEC/D3) under inflammatory conditions. Results. 95% of the clones common to EAE and HEALTHY repertoires were subtracted, allowing clone enrichment. Conclusion. This physical subtraction discarded from a complex repertoire the non-specific selected ligands. STRATEGY 1) Three rounds of in vivo phage peptide selection in EAE female Lewis rats ("EAE repertoire") vs controls ("HEALTHY repertoire"). 2) DNA subtraction of the most common sequences between «HEALTHY» and «EAE» phage repertoires to obtain a third EAE specific «SUBTRACTION » phage repertoire. 3) Massive sequencing of the three repertoires and bioinformatic analysis to identify the peptides sequences with high EAE specificity. 4) Biological tests of potential EAE specific phage clones with CNS tissues from EAE and Healthy control rats. 5) Biological tests of the EAE specific peptide and phage clones on the BBB in vitro model (hCMEC/D3 cells) under inflammatory conditions (IL-1β stimulation). 6) Target separation and identification by cross-link between the selected phage clones and hMEC/D3 endothelial cells targets under IL-1β stimulation vs controls.
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