Generation of novel anti-BTLA monoclonal antibodies for in vivo use and their functional testing at near-physiological conditions.

We have generated and characterized a panel of monoclonal antibodies recognizing B and T lymphocyte attenuator (BTLA), a transmembrane protein expressed on essentially all lymphoid cells. One of the monoclonal antibodies (MAbs) detects for the first time all BTLA protein variants described for various mouse strains with high sensitivity, both in flow cytometry and immunohistology. Further tests have determined that this MAb recognizes a BTLA epitope independent of the HVEM binding site. Moreover, we identified a number of antibodies capable of efficiently blocking the interaction of BTLA with its ligand herpes virus entry mediator (HVEM). A series of experiments was performed with these MAbs at near-physiological conditions to assess their blocking potential in vivo. These tests, performed with whole MAbs and also their F(ab)(2) formats, revealed that measurements of binding at 37 degrees C to primary cells expressing the target protein on the cell surface offer superior information on their blocking capacity. The generated BTLA-specific MAb will be used for in vivo studies to further elucidate the biological role of BTLA-HVEM interaction and function in vivo.