C. Miyazaki et al., Changes in the specificity of antibodies by site-specific mutagenesis followed by random mutagenesis, PROTEIN ENG, 12(5), 1999, pp. 407-415
The specificity for 11-deoxycortisol (11-DOC) of a monoclonal antibody (mAb
), designated SCET, was changed to specificity for cortisol (CS) by site-sp
ecific mutagenesis followed by random mutagenesis. The Fab form of SCET was
expressed on the surface of a phage, During the first step, mutations were
introduced at 14 amino acid positions in three complementarity-determining
regions (CDRs) of the VH domain that seemed likely to form the steroid-bin
ding pocket. A clone, DcC16, was isolated from the resultant library with m
ultiple mutations and this clone was shown to have CS-binding activity but
also to retain high 11-DOC-binding activity. During the second step, mutati
ons were introduced randomly into the entire V-H- coding region of the DcC1
6 clone by an error-prone polymerase chain reaction, and CS-specific mutant
antibodies were selected in the presence of 11-DOC as a competitor. Three
representative clones were analyzed with the BIAcore instrument, and each r
evealed a large increase in the binding constant for CS and a decrease in t
hat for 11-DOC. Structural models, constructed by computer simulation, indi
cated the probable molecular basis for these changes in specificity.