Kansas Biosciences Authority from Joint Call (KBA funding only)
ASFVp30, p54, p72, and CD-2 like protein epitope mapping and development of blocking and antigen detection assays (KBA Funding Only)
Kansas State University PI: Raymond Rowland
USDA-APHIS Co-I: Wei Jia
07/01/2015 – 06/30/2016
Abstract:
The development of diagnostic tests for the detection of transboundary animal diseases is among the priorities established by the NPB and CEEZAD. At present, diagnostic tests for the detection of ASFV antibodies or antigens are not commercially available in the US. This is an important deficit in the mission to protect US agriculture. While tests made outside the US may possess good sensitivity and specificity, a lack of transparency when test modifications are implemented is of concern to US stakeholders. Another concern among stakeholders is the lack of availability of tests that are sufficiently sensitive to rapidly detect a new introduction of virus and, at the same time, not yield false-positive results, which could shutdown America’s pork exports. And finally, stakeholders are keenly interested in having a test that can be easily adapted for use in pen-side, as well as high throughput applications.
The overall objective of this project is to support stakeholder needs by producing ASF reagents and assays for use in the US and other countries, and to improve the sensitivity and specificity of existing tests. This project is a continuation of an ongoing collaboration between the Foreign Animal Disease Diagnostic Laboratory and Rowland laboratory. With previous support from DHS, FADDL produced a panel of 161 monoclonal antibodies (mAbs) against four important ASFV structural proteins: p30 (22 mAbs), p72 (29 mAbs), p54 (12 mAbs), and CD-2 like protein (13 mAbs). For this proposal, the mAbs are used by the Rowland lab to identify specific epitopes that each mAb recognizes. Candidate mAbs will be used to develop two types of antibody-based assays. The first is a blocking or competitive ELISA. In this format, a mAb specific for an epitope is blocked or displaced by antibodies present in the serum. For detection of FAD, this preferred format offers maximum specificity while avoiding false positive results. The second type of test is the antigen capture ELISA. In this format, one mAb captures the ASFV antigen of interest and a second mAb detects the antigen creating a sandwich. Antigen capture is used when serum antibodies are not present and offers a good backup to serological assays. For this test it is important to identify two mAbs with high affinity that recognize different epitopes. And finally, mAb are used to detect ASFV in western blots on samples from infected animals or cell culture. These will be valuable research tools. The actual field validation and commercialization of the blocking and antigen capture tests is beyond the scope and timeline of the grant and will be the subject of future funding requests.
Co-detection of different genotypes of FMDv, CSFv and ASFv with a high-throughput MassTag system (KBA Funding Only)
Kansas State University PI: Jianfa Bai
Kansas State University Co-I: Ying Fang
Kansas State University Co-I: Xuming Liu
Kansas State University Co-I: Gary Anderson
07/01/2015 – 06/30/2016
Abstract:
The objective of this research proposal is to develop a high throughput pathogen identification system using the MassTag platform for the detection and genotype differentiation of foot-and-mouth disease virus (FMDv), African swine fever virus (ASFv) and classical swine fever virus (CSFv). This proposed study does not overlap with the joint effort of Lawrence Livermore National Laboratory and KSU for the development and validation of a multiplex assay to simultaneously diagnose multiple swine respiratory diseases in a single sample, which includes major respiratory pathogens, but does not involve genotyping work on foreign animal diseases (FADs); this CEEZAD proposal is aimed to detect and differentiate different genotypes of FMDv, ASFv, and CSFv, providing information about potential FAD threat that relates to potential introduction sources of the pathogens and their geographic distribution of a specific genotype.