Isolation and characterization of novel single-chain Fv specific for human granzyme B

Abstract

Granzyme B, a neutral serine protease, has been demonstrated to be a pivotal molecule for protective immunity against viral infection and cellular malignant transformation. To facilitate monitoring of granzyme B levels, we have recently applied phage display technology to produce single-chain Fv antibodies specific for granzyme B, as versatile alternatives and complementary reagents to currently available monoclonal antibodies. Through four rounds of panning on purified human granzyme B-coated on solid phase, three unique clones were isolated. Expressed soluble scFv antibodies demonstrated specific immunological applications including ELISA, Western blotting, immunoprecipitation and intracellular staining. Based on sequence analyses and structural modeling, one scFv, Fv17, may have overlapping antigen binding specificity with monoclonal antibodies 2C5/F5 and GB11. Owing to the availability of its DNA sequence and large scale production capability, Fv17 should be a superior reagent for monitoring granzyme B expression in natural killer cells and antigen specific CD8+ T cell immunity.