Two-step in vitro antibody affinity maturation enables estradiol-17beta assays with more than 10-fold higher sensitivity. 2010

Norihiro Kobayashi, and Hiroyuki Oyama, and Yoshinori Kato, and Junichi Goto, and Eskil Söderlind, and Carl A K Borrebaeck
Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan. no-kobay@kobepharma-u.ac.jp

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.

UI MeSH Term Description Entries
D007135 Immunoglobulin Variable Region That region of the immunoglobulin molecule that varies in its amino acid sequence and composition, and comprises the binding site for a specific antigen. It is located at the N-terminus of the Fab fragment of the immunoglobulin. It includes hypervariable regions (COMPLEMENTARITY DETERMINING REGIONS) and framework regions. Variable Region, Ig,Variable Region, Immunoglobulin,Framework Region, Immunoglobulin,Fv Antibody Fragments,Fv Fragments,Ig Framework Region,Ig Variable Region,Immunoglobulin Framework Region,Immunoglobulin Fv Fragments,Immunoglobulin V,Antibody Fragment, Fv,Antibody Fragments, Fv,Fragment, Fv,Fragment, Fv Antibody,Fragment, Immunoglobulin Fv,Fragments, Fv,Fragments, Fv Antibody,Fragments, Immunoglobulin Fv,Framework Region, Ig,Framework Regions, Ig,Framework Regions, Immunoglobulin,Fv Antibody Fragment,Fv Fragment,Fv Fragment, Immunoglobulin,Fv Fragments, Immunoglobulin,Ig Framework Regions,Ig Variable Regions,Immunoglobulin Framework Regions,Immunoglobulin Fv Fragment,Immunoglobulin Variable Regions,Regions, Immunoglobulin Variable,Variable Regions, Ig,Variable Regions, Immunoglobulin
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D011863 Radioimmunoassay Classic quantitative assay for detection of antigen-antibody reactions using a radioactively labeled substance (radioligand) either directly or indirectly to measure the binding of the unlabeled substance to a specific antibody or other receptor system. Non-immunogenic substances (e.g., haptens) can be measured if coupled to larger carrier proteins (e.g., bovine gamma-globulin or human serum albumin) capable of inducing antibody formation. Radioimmunoassays
D003429 Cross Reactions Serological reactions in which an antiserum against one antigen reacts with a non-identical but closely related antigen. Cross Reaction,Reaction, Cross,Reactions, Cross
D004958 Estradiol The 17-beta-isomer of estradiol, an aromatized C18 steroid with hydroxyl group at 3-beta- and 17-beta-position. Estradiol-17-beta is the most potent form of mammalian estrogenic steroids. 17 beta-Estradiol,Estradiol-17 beta,Oestradiol,17 beta-Oestradiol,Aerodiol,Delestrogen,Estrace,Estraderm TTS,Estradiol Anhydrous,Estradiol Hemihydrate,Estradiol Hemihydrate, (17 alpha)-Isomer,Estradiol Monohydrate,Estradiol Valerate,Estradiol Valeriante,Estradiol, (+-)-Isomer,Estradiol, (-)-Isomer,Estradiol, (16 alpha,17 alpha)-Isomer,Estradiol, (16 alpha,17 beta)-Isomer,Estradiol, (17-alpha)-Isomer,Estradiol, (8 alpha,17 beta)-(+-)-Isomer,Estradiol, (8 alpha,17 beta)-Isomer,Estradiol, (9 beta,17 alpha)-Isomer,Estradiol, (9 beta,17 beta)-Isomer,Estradiol, Monosodium Salt,Estradiol, Sodium Salt,Estradiol-17 alpha,Estradiol-17beta,Ovocyclin,Progynon-Depot,Progynova,Vivelle,17 beta Estradiol,17 beta Oestradiol,Estradiol 17 alpha,Estradiol 17 beta,Estradiol 17beta,Progynon Depot
D005818 Genetic Engineering Directed modification of the gene complement of a living organism by such techniques as altering the DNA, substituting genetic material by means of a virus, transplanting whole nuclei, transplanting cell hybrids, etc. Genetic Intervention,Engineering, Genetic,Intervention, Genetic,Genetic Interventions,Interventions, Genetic
D006241 Haptens Small antigenic determinants capable of eliciting an immune response only when coupled to a carrier. Haptens bind to antibodies but by themselves cannot elicit an antibody response. Hapten,Contact-Sensitizing Agents,Agents, Contact-Sensitizing,Contact Sensitizing Agents
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA

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