Location: Foreign Animal Disease Research
2012 Annual Report
The specific objectives are: 1. Produce paired monoclonal antibodies to porcine cytokines IL-13 and IL-15. 2. Produce paired mononclonal antibodies to bovine cytokines IL-13, IL-15, IL-17 and IL-21. 3. Produce a collection of monoclonal antibodies that will differentially bind alleles of the BoLA Class I and Class II major histocompatibility complex (MHC) proteins that can be used to rapidly analyze blood samples and determine MHC gene expression in a given cow.
Amendment 1: 4. Produce monoclonal antibodies to porcine immunoglobulins.
In FY 2012 a virus vector, replication defective human Adenovirus 5 (Ad5) was constructed to express the bovine cytokine, interleukin (IL)-13. This Ad5-bIL13 virus was inoculated into three mice and the animals boosted with virus vector expressing bIL-13 and test-bled. Anti-sera from these mice are currently being screened for presence of antibody specific for bIL-13.
Much of the work at Green Mountain Antibody, Inc. thus far has focused on the vector Ad5-bIL13FLAG. This construct will be used to generate purified bovine cytokine via the FLAG-tag and that protein is subsequently used for detection of anti-bIL13 antibodies in mouse serum or hybridoma supernatants. A number of cell lines were screened for their ability to express the cytokine following infection with Ad5-bIL13 FLAG. Following screening and consultation with ARS, PIADC, one cell line was selected for protein expression: Madin-Darby Bovine Kidney cells (MDBK). A procedure consisting of immunoprecipitation with mouse anti- FLAG antibody and western blot with rabbit anti-Flag antibody has been used to successfully detect IL-13 FLAG in MDBK supernatants and lysates. Western blot assays were then performed to estimate the protein expression.
Technologies developed: The goal of this collaboration is to create a series of monoclonal antibodies to bovine and porcine cytokines. These antibodies are to be used by researchers ARS, PIADC. The estimated timeline to have the first set up antibodies is by the end of 2013.
Publications: No publications have been produced to date.