The anti-glutathione antibody scFv 20C9, which we previously isolated from a human synthetic phage antibody scFv library [Hirose, M., Hayano, T., Shirai, H., Nakamura, H., and Kikuchi, M. (1998) Protein Eng. 11, 243-248], was expressed in the E. coli pET system and purified by sequential chromatography on Ni and glutathione-conjugated affinity resins. The purified scFv 20C9 antibody was characterized for its binding affinity for several glutathione derivatives by the BIACORE system, Although GSH, GSSG, and gamma -Glu-Cys could bind to the immobilized antibody, this was not the case for Cys-Gly, L-Glu, L-Cys, L-Gly, or several other glutathione derivatives such as gamma -Glu-Ser-Gly. The results suggest that a gamma -glutamic acid and sulfur atom are important for scFv 20C9 antibody recognition of glutathione. This is the first report to indicate that an scFv antibody can recognize a region as small as a dipeptide. (C) zool Academic Press.