REAGENTS FOR IMMUNOLOGIC ANALYSIS OF COTTON RAT.

Cotton rat S.hispidus is a highly translational model of human infectious diseases that has been used successfully to demonstrate efficacy of several human vaccines and therapeutics. Yet, few reagents are available for the evaluation of immune correlates of protection and assessing immunopathology of disease in cotton rats. In this work, we will develop monoclonal antibodies for identification and tracking of various types of lymphocytes and granulocytes, as well as antibodies against different IgG subtypes and Fc gamma receptors in S.hispidus. Target cotton rat proteins (20 total) will be expressed in human 293 cells and used to immunize BALB/c mice for monoclonal antibody (mAb) production. mAbs will be characterized for binding to recombinant and/or native protein using flow-cytometry, ELISA, and Western blot. Knock-in cells will be generated using human 293T or cotton rat CCRT cells transfected to express proteins of interest. To confirm antibody specificity, competition assays blocking antibody binding to native cotton rat antigens (isolated from cotton rat tissues) will be performed using recombinant antigens. Affinity of antibodies will be determined by Biolayer Interferometry (BLI) assay. mAbs against immunologic targets generated in this Phase I project will be ready for further characterization and optimization in the future Phase II work.