Location: Foreign Animal Disease Research2012 Annual Report
1a. Objectives (from AD-416):
The objective of this agreement is to develop a number of monoclonal antibodies specific for T cell derived cytokines in cattle and swine and to develop a series of monoclonal antibodies to bovine leukocyte antigen (BoLA) gene products in order to develop a rapid method to tissue type cattle. 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.
1b. Approach (from AD-416):
ARS, PIADC and Green Mountain Antibodies will exchange reagents and produce monoclonal antibodies to cattle and swine proteins. Genes encoded with specific proteins will be cloned into the replication defective human adenovirus 5 vector (huAd5). Animal trials (mice, rabbit, cattle and swine) will be conducted and subsequent protein analysis conducted. Mice will then be immunized with Ad5 vectors expressing the protein. Mice will be tested for production of antibody reactive with the protein and positive mice will be tested for antibody production. In vitro screening will be conducted and the proteins generated will be purified.
3. Progress Report:
The goal of this collaborative agreement, which was initiated in FY 2012, is to produce monoclonal antibodies to a series of bovine and porcine cytokines. These monoclonal antibodies are not commercially available and will be valuable tools to study bovine and porcine immune responses to foot and mouth disease challenge. Historically, monoclonal antibody production at Green Mountain Antibody has been done via antigen immunization of mice and rats. The collaboration with ARS, PIADC requires the development of methods to use adenovirus vectors as methods to deliver immunogens to mice. These adenovirus constructs were expanded, purified, and titrated. New methods are being developed to screen antisera and hybridomas producing antibodies against these cytokines. 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.