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B and T lymphocyte attenuator (BTLA) is recently identified as the third co-inhibitory receptor with similarities to CTLA-4 and PD-1. Previous reports have shown that BTLA is associated with autoimmune diseases, viral infections and tumor immune evasion. However, the possibility of the existence and role of a soluble form of human BTLA (sBTLA) has not been revealed. Based on our previously generated mouse anti-BTLA monoclonal antibodies, we intend here to develop a novel enzyme-linked immunosorbent assay (ELISA) for detecting sBTLA. Using monoclonal (MAb) 8H9 as coated antibody and the biotin-labeled MAb 7D7 for detection, a sandwich ELISA was developed with good sensibility, line reliability, and specificity. With the established ELISA, the existence and concentration of sBTLA were demonstrated for the first time. It was found that soluble sBTLA existed in the sera of healthy donors and the quantitation of sBTLA climbed along with increasing age, indicating sBTLA may be correlated with immune dysregulation resulting from the aging. Hence, a sandwich ELISA for detecting sBTLA was successfully established and soluble BTLA existing in human serum was first identified, which might play an important role in immunoregulation.

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.