Two-Step in Vitro Antibody Affinity Maturation Enables Estradiol-17β Assays with More than 10-Fold Higher Sensitivity

摘要

Immunoassays for haptens depend on competitive hapten-nanti-hapten reactions, and consequently their sensitivitiesnare significantly influenced by the affinities of antihaptennantibodies. Thus, genetically engineered antibodies,nwhich have much higher affinities than native antibodies,nshould increase assay sensitivities. Here, we created anmutated single-chain Fv fragment (scFv) against estradiol-n17u0001 (E2) that allowed immunoassays with a muchnimproved sensitivity. Two steps of affinity maturationnwere performed on a “wild-type” scFv (scFv#E4-4)ncomposed of VH and VL domains from a mouse anti-E2nantibody (Ab#E4-4). First, we conducted complementarity-ndetermining region (CDR)-targeted mutagenesisnby “CDR-shuffling”. Gene fragments encoding CDRsnH2, H3, L1, and L3, each of which contained randomnpoint mutations, were combined by “shuffling” into thengene encoding the scFv#E4-4 scaffold. After phagendisplay and repeated panning, we isolated a mutatednscFv clone [scFv#m1-e7; IleL29Val] that had 5-foldnhigher affinity (Ka ) 2.6 × 108 M-1) compared to thenAb#E4-4 Fab fragment (Fab#E4-4). Next, the entire VHnand VL of this clone were randomly mutated by errorpronenpolymerase chain reaction (PCR). From thisnlibrary, we found an improved clone, scFv#m2-c4 (Kan) 6.3 × 108 M-1; LysH19Arg, TyrH56Phe, SerH84Pro,nGluH85Gly, GlnL27Arg, LeuL36Met, SerL63Gly, andnSerL77Gly). ScFv#m2-c4 had more than 10-fold highernsensitivity (the midpoint of its dose-response curvenwas 0.56 ng) than Fab#E4-4 (midpoint 9.0 ng/assay)nin a competitive E2 radioimmunoassay, and evennhigher sensitivity [midpoint 21 pg/assay, and a limitnof detection of 0.47 pg (1.7 fmol)/assay] in a competitivenenzyme-linked immunosorbent assay. Crossreactivitynwith selected E2-related endogenous steroidsnstrongly suggested that scFv#m2-c4 has improvednspecificity compared to conventional antibodies.