We propose to develop a rapid lateral flow antigen capture assay that can reliably detect exposure to, or infection with, Yersinia pestis or genetically modified Yersinia based bioweapons. There is a need for a reliable rapid assay to be used by minimally trained personnel under virtually any condition. A rapid assay that detects the Y. pestis F1 capsular antigen has been developed but that assay cannot be depended on in case of a biological attack because the F1 antigen is not required for virulence. To overcome the current assay, an enemy could simply use fully virulent Y. pestis lacking the F1 capsular antigen. A new assay that captures one or more required virulence factors in addition to the F1 antigen would not have this problem. We selected five antigens in addition to F1 as potential capture targets: Low calcium response V protein (LcrV), plasminogen activator protease (Pla) and YopM, YopD and YopH. By adding one or more of these antigens in addition to F1 as a target for capture we will resolve the problem associated with the current assay. Our final state-of-the-art assay will be able to discriminate between F1 positive and F1 negative virulent Yersinia pestis and will be able to distinguish Y. pestis from other species of Yersinia (Y. pseudotuberculosis and Y. enterocofitica). As proof-of-principle, we developed two LcrV-capture prototype assays, an ELISA and a Lateral-flow assay, using monoclonal antibodies to both capture and detect the antigen. In vitro studies suggest that during early infection, LcrV concentrations range between 20 and 500ng/ml. The LcrV-capture ELISA has a detection limit threshold of about 0.1ng/ml, and in a preliminary experiment, the lateral-flow assay detected the lowest concentration of LcrV used (l.5ng/ml). In addition, we developed a specimen pre-treatment protocol and successfully used it to detect secreted native Y. pestis LcrV added to sputum. These results indicate that the specimen pre-treatment protocol was effective in liberating LcrV for capture. It detected the lowest concentration of LcrV tested on the clinical samples. We will further optimize both the ELISA and the rapid lateral flow antigen capture assay. Then, we will develop rapid assays that detect multiple Y. pestis antigens. As a final step, we will select the best system and optimize it to a final rapid antigen capture assay. The ultimate goal is to develop a highly sensitive multi-antigen rapid lateral flow (or dipstick) assay that will detect levels as low as 0.1ng/ml of Y. pestis antigen.
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