title: Two-step in vitro antibody affinity maturation enables estradiol-17beta assays with more than 10-fold higher sensitivity.
abstract: 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-17beta (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(L29)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.
authors.affiliation: Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan. no-kobay@kobepharma-u.ac.jp
authors.forename: Norihiro
authors.initials: N
authors.lastname: Kobayashi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Oyama
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kato
authors.forename: Junichi
authors.initials: J
authors.lastname: Goto
authors.forename: Eskil
authors.initials: E
authors.lastname: Söderlind
authors.forename: Carl A K
authors.initials: CA
authors.lastname: Borrebaeck
mesh_terms: D000818:Animals; D001483:Base Sequence; D003429:Cross Reactions; D004958:Estradiol / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D005818:Genetic Engineering; D006241:Haptens / Q000378:metabolism; D007135:Immunoglobulin Variable Region; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D011863:Radioimmunoassay / Q000379:methods*; D012680:Sensitivity and Specificity; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D006241:Haptens; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D057127:Single-Chain Antibodies; D004958:Estradiol