Two-step in vitro antibody affinity maturation enables estradiol-17[beta] 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.sub.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.sub.H] and [V.sub.L] domains from a mouse anti-[E.sub.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#ml-e7; [Ile.sup.L29]Val] that had 5-fold higher affinity ([K.sub.a] = 2.6 x [10.sup.8] [M.sup.-1]) compared to the Ab#E4-4 Fab fragment (Fab#E4-4). Next, the entire [V.sub.H] and [V.sub.L] of this clone were randomly mutated by errorprone polymerase chain reaction (PCR). From this library, we found an improved clone, scFv#m2-c4 ([K.sub.a] = 6.3 x [10.sup.8] [M.sup.-1]; [Lys.sup.H19]Arg, [Tyr.sup.H56]Phe, [Ser.sup.H84]Pro, [Glu.sup.H85]Gly, [Gln.sup.L27]Arg, [Leu.sup.L36]Met, [Ser.sup.L63]Gly, and [Ser.sup.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.sub.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. Crossreactivity with selected [E.sub.2]-related endogenous steroids strongly suggested that scFv#m2-c4 has improved specificity compared to conventional antibodies. 10.1021/ac902283n