Oral Presentation International Veterinary Immunology Symposium 2016

Monoclonal antibodies for the analysis of bovine CD4+ helper T cell subsets (#45)

Tatjana Sitt 1 , Rebecca Calder 1 2 3 , Jared R Patch 1 2 , Mary A Kenney 2 , Jenna Khan 3 , Ellie Bouffard 3 , Amy Griffiths 3 , John W Barlow 1 , William Church 3 , William T Golde 2
  1. Department of Animal and Veterinary Sciences, University of Vermont, Burlington, VT, United States
  2. Plum Island Animal Disease Center, Agricultural Research Service, USDA, Greenport, New York, USA
  3. Green Mountain Antibodies, Inc., Burlington, VT, USA

Veterinary immunology and infectious disease research is at the interface of human and animal health (food security, zoonotic disease). Reagents for analysis of immune responses in veterinary species are critical for understanding disease pathogenesis, vaccine performance and immunologic vigor. However, knowledge of disease pathology and immune responses in cattle continues to be hindered by the paucity of validated monoclonal antibodies (mAb) to immune markers. Such reagents, though ample for human and mice, are limited for livestock species. Efforts have been made to increase the quality, quantity and availability of well-defined mAb to researchers through multiple initiatives resulting in a steadily expanding, yet incomplete, antibody collection. Well characterized bovine antibodies for identification of TH2 cells (IL-4 and IL-13) and TH17 cells (IL-17A, IL-17F, IL-21 and IL-22) were lacking. Thus, IL-13 and IL-17A antibodies were generated using mice immunized with replication defective Adenovirus5 vectors (Ad5) expressing the bovine cytokines IL-13 and IL-17A. Mouse spleen cells were fused with a murine myeloma fusion partner to produce IL-13 and IL-17A antibodies. Recombinant cytokine antigens were generated in vitro and used for hybridoma screening and antibody characterization. Hybridoma produced and purified mAbs were characterized in western blots, ELISA and intracellular cytokine staining (ICS) using flow cytometry. Clones of antibodies having similar antigen binding properties were further characterized by PCR amplification and sequencing of light and heavy chain variable regions. Using these new mAbs in combination with available bovine specific antibodies to IL-4 and IFNγ, we are developing multicolor flow analysis for the further characterization of antigen specific, CD4+ helper T cell subset responses in cattle. This approach will lead to increased understanding of infectious disease pathology, vaccine efficacy, and immunologic vigor allowing for more effective vaccine design, development and clinical testing. Future work will also include production, testing, and validation of IL-17F, IL-21 and IL-22.