The need for nimble technology platforms to counter bioterrorism threats motivates the research proposed here. In this project, reagents with high affinity and specificity for vaccinia virus proteins will be selected from phage-displayed libraries. The ultimate goal is to identify a specific, high affinity binding partner, or """"""""Artificial Antibody"""""""" (ArtAb), for each protein in the vaccinia virus (smallpox vaccine) proteome. The approach proposed here to accomplish this applies vast libraries of novel protein variants fused to filamentous phage particles (""""""""phage display""""""""). Libraries will apply ankyrin and leucine-rich repeat scaffolds with vast diversity (>10[11] different molecules). Various phage-display techniques, including some schemes invented by G. Weiss (Co-Investigator), will be used to hone binding affinity and specificity of vaccinia protein binding partners. In order to select phage particles encoding specific binding partners against each individual vaccinia virus protein, it will be necessary to generate each of the 266 viral proteins to be used as targets for panning. For this purpose a high throughput protein expression approach developed by the investigator called """"""""PCR Express"""""""" will be employed. PCR Express combines PCR cloning and in vitro transcription/translation to rapidly generate 6xHis and HA tagged proteins from their PCR amplified genes. The individual proteins will be coated onto separate wells of either nickel, or anti-HA coated 96-well plates, and the plates will be used to scan the phage display libraries for high affinity, vaccinia virus phage particles encoding protein specific Artificial Antibodies (ArtAbs). The sequences encoding each ArtAb will be PCR amplified from the selected phage, and PCR Express will be used to synthesize each ArtAb protein from its PCR product. This will result in an array of protein specific binders against each protein in the vaccinia virus proteome to produce the synthetic anti-proteome array. """"""""Proof of concept"""""""" demonstrations to show the utility of this artificial antibody array in several analytical assays will be investigated. HeLa cells will be infected with vaccinia virus and the temporal expression level of each of the vaccinia virus proteins will be followed using an """"""""ELISA type"""""""" and Western blot assays. The feasibility of using BIAcore angular reflection for quantifying protein binding to anti-proteome microarray chips will be investigated. The artificial antibodies will be evaluated for in situ temporal and special localization in sections from vaccinia virus infected mouse lungs. Successful """"""""proof of concept"""""""" demonstrations in any of these assays will form the basis for a Phase II application leading, to arrays of analytical proteomics reagents against the other Class A, B & C bioterrorism agents.
