The overall objective of this Fast-Track project is to develop a Rapid-format immunodiagnostic assay capable of quick detection of contamination or infection with Yersinia pathogens, especially biologically engineered pathogens that might be used as weapons of mass destruction or as agents of bioterrorism. The primary target product is a lateral flow (Rapid-format) cassette using an antigen-capture strategy. The antigen that we propose to capture is the LcrV protein, which is absolutely required for virulence and is known to contain several strongly antigenic regions whose sequences are highly conserved across all known strains of the pathogenic Yersinia species. The applicant research group is experienced in the conception and development of Rapid-format immunodiagnostic assays. One of these assays, developed under an NIH SBIR grant is currently in the market. Existing immunodiagnostic assays for Yersinia pestis are designed for use in the context of natural pathogens in natural epidemics. They lack certain characteristics that are fundamental to an assay to be used under field conditions to detect contamination or infection with genetically optimized bioweapons. In Preliminary Studies we have designed an antigen capture immunoassay based on highly conserved antigenic segments of the LcrV protein, which is required for virulence in all of the Yersinia pathogens. In this Phase I project, monoclonal antibodies will be developed against recombinant LcrV protein and against synthetic peptides covering the highly conserved antigenic regions of LcrV. These monoclonal antibodies will be tested both on western blot and in an enzyme-linked immunoassay. In the accompanying Phase II proposal, these reagents will be used to create an antigen-capture immunoassay based on a virulence antigen, which cannot be deleted from a biological weapon or modified to circumvent the assay. Furthermore, this prototype assay will be reduced to a production model, validated for regulatory approval, and finalized for manufacture through a joint venture partner.