1. CSF is a highly contagious viral disease of swine. 2. The US is free of this disease; however, the disease is widespread in most islands of the Caribbean Sea, and in Mexico, and Central and South America. 3. Either enhancement of presently available tests or development of newer tests is of utmost importance. 4. The US must be prepared to protect its swine industry from the threat of either inadvertent or deliberate introduction of CSF. 1. Our proposal is to make cheaper recombinant antibodies for the eventual development of a pen-side antigen capture assay. A sufficiently cheap pen-side test for more extensive use in surveillance of inadvertent or deliberate introduction of diseases will be very useful for risk management of emerging diseases and as a biosecurity measure at the farm level. 2. In order to create a pen-side assay for the detection of CSFV, OmniSite proposes the development of a hand-held, lightweight, easy-to-use detection system to identify the target of interest using pre-loaded reagents in a microelectromechanical system (MEMS) cartridge. OBJECTIVES: The overall objective of this project is to develop an integrated biosensor system that can be used to monitor the health status of animals, and enabling managers to identify infected animals for early removal prior to disease spread. OmniSite BioDiagnostics, Inc. (OmniSite) proposes to develop a Handheld MEMS (microelectromechanical) Point Detection Sensor employing a magnetic bead-based assay for the diagnosis of CSF. The sensor will contain bivalent recombinant antibody molecules specific for the E2 and Erns for capture of CSFV from tissue and fecal/manure samples. APPROACH: OmniSites objective is to develop bivalent recombinant antibody molecules specific for components of the Classical Swine Fever virus, specifically the envelope glycoproteins E2 and Erns and begin prototyping a pen-side diagnostic assay. Phase I tasks will be divided between the device prototype work to be completed at OmniSite. OmniSite will sub-contract jointly with Drs. G. Gale Wagner and Surya Waghela of Texas A&M University, College Station, Texas, Department of Veterinary Pathobiology and Dr. Antonio Morilla, Head of Swine Infectious Disease Branch, Centro Nacional de Microbiologia, Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias (INIFAP), Mexico, for the development of specific recombinant antibodies to CSFV. It will be necessary to complete the primary antibody work in Mexico due to restrictions on use of the virus in the United States, which is currently free of CSF. 1. Select anti-E2 and anti-Erns scFv from a paratope library of immunoglobulin VH and VL genes prepared from lymphocytes of mice immunized with the E2 and Erns antigen. 2. Engineering and expression of the bivalent anti-E2 and anti-Erns sc(Fv)2. ii) Evaluate the effectiveness of sc(Fv)2 in capturing CSFV. 3. Detecting the captured CSFV with anti-Erns sc(Fv)2. 4. Field trials with the proposed test. 5. Prototype a MEMS cartridge for testing. 6. Conjugate the recombinant antibodies to magnetic microbeads and to fluorescein and demonstrate binding in an assay format. 7. Begin microfluidic optimization in the prototype MEMS cartridge
