Altered protein processing, including misfolding and aggregation, is a pathological aspect of numerous neurodegenerative diseases including Alzheimers, Parkinsons and related synucleionopathies, and Huntingtons Disease (HD). Aggregation of the protein huntingtin (htt) into fibrils has been associated with HD. The exon 1 portion of htt has been shown to form aggregates in vitro and in vivo. Htt can form a number of different morphologies including branched fibrils and buds. The roles of the various morphologies in the progression of HD are not known. Here we demonstrate a novel technique that can be used to isolate morphology specific antibodies expressed on the surface of bacteriophage from a nave phage display antibody library by panning against oligomeric forms of the protein alpha-synuclein. Alpha-synuclein was chosen as a model protein to develop this protocol since it aggregates more slowly than htt. Our results demonstrate that phage displayed antibodies against the target protein can be isolated after only two rounds of selection. Moreover, using this AFM based technique we can isolate antibodies to the oligomeric morphology of alpha-synuclein. After only 2 rounds of selection, 20 out of 48 clones showed positive binding as indicated by ELISA analysis, and 11of the 20 positive clones contained full length scFv as indicated by PCR. The specificity of the selected scFv-phages against oligomeric alpha-synuclein was verified by Atomic Force Microscopy.
|Original language||English (US)|
|State||Published - May 10 2006|