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

Immunoassays for haptens depend on competitive hapten-anti-hapten reactions, and consequently their sensitivities are significantly influenced by the affinities of anti-hapten antibodies. Thus, genetically engineered antibodies, which have much higher affinities than native antibodies, should increase assay sensitivities. Here, we created a mutated single-chain Fv fragment (scFv) against estradiol-17 beta (E-2) that allowed immunoassays with a much improved sensitivity. Two steps of affinity maturation were performed on a wild-type scFv (scFv#E4-4) composed Of V-H and V-L domains from a mouse anti-E-2 antibody (Ab#E4-4). First, we conducted complementarity-determining region (CDR)-targeted mutagenesis by CDR-shuffling. Gene fragments encoding CDRs H2, H3, L1, and L3, each of which contained random point mutations, were combined by shuffling into the gene encoding the scFv#E4-4 scaffold. After phage display and repeated panning, we isolated a mutated scFv clone [scFv#m1-e7; Ile(1,29)Val] that had 5-fold higher affinity (K-a = 2.6 x 10(8) M-1) compared to the Ab#E4-4 Fab fragment (Fab#E4-4). Next, the entire V-H and V-L of this clone were randomly mutated by error-prone polymerase chain reaction (PCR). From this library, we found an improved clone, scFv#m2-c4 (K-a = 6.3 x 10(8) M-1; Lys(H19)Arg, Tyr(H56)Phe, Ser(H84)Pro, Glu(H85)Gly, Gln(L27)Arg, Leu(L36)Met, Ser(L63)Gly, and Ser(L77)Gly). ScFv#m2-c4 had more than 10-fold higher sensitivity (the midpoint of its dose-response curve was 0.56 ng) than Fab#E4-4 (midpoint 9.0 ng/assay) in a competitive E-2 radioimmunoassay, and even higher sensitivity [midpoint 21 pg/assay, and a limit of detection of 0.47 pg (1.7 fmol)/assay] in a competitive enzyme-linked immunosorbent assay. Cross-reactivity with selected E-2-related endogenous steroids strongly suggested that scFv#m2-c4 has improved specificity compared to conventional antibodies.