title: One-Shot In Vitro Evolution Generated an Antibody Fragment for Testing Urinary Cotinine with More Than 40-Fold Enhanced Affinity.
abstract: Immunoassays for cotinine, a major nicotine metabolite, in the urine are useful for monitoring the degree of tobacco smoke exposure. However, hybridoma-based anti-cotinine antibodies lack sufficient binding affinity to perform practically sensitive measurements, and thus most cotinine assays still rely on polyclonal antibodies. Here, we describe the generation of a mutant single-chain Fv fragment (scFv) that was used in an enzyme-linked immunosorbent assay (ELISA) to determine urinary cotinine levels in passive smokers. A "wild-type" scFv (scFv-wt) with a Kavalue of 2.7 × 107M-1(at 4 °C) was prepared by linking the VHand VLdomains in a mouse anti-cotinine antibody. "One-shot" random mutagenesis on the scFv-wt gene by error-prone PCR generated mutant scFv genes, which were expressed on phage particles. Repeated panning directed toward mutants with slower off-rates selected scFv clones that showed improved sensitivity in an ELISA system. One of these mutants (scFv#m1-54) with five amino acid substitutions showed more than a 40-fold enhanced Ka(1.2 × 109M-1at 4 °C) and, thus, was used to monitor human urinary cotinine. A limited amount of soluble scFv was reacted with urine specimens (or cotinine standards) at 4 °C for 120 min in microwells on which cotinine residues had been immobilized. The midpoint of the dose-response curves under optimized conditions (0.27 ng/assay) was more than 100-fold lower than the ELISA results obtained using scFv-wt. The limit of detection (8.4 pg/assay) corresponded to 0.17 ng/mL urinary cotinine, which was satisfactorily low for testing the threshold levels for passive smoke exposure. The assay values for volunteers correlated with the values determined using a commercial assay kit. This study evidently showed the potential of a molecular breeding approach, in which simple in vitro evolution might generate superior antibody reagents as cloned proteins, overcoming the limited molecular diversity inherent to conventional immunization-based antibodies.
authors.affiliation: Kobe Pharmaceutical University , 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Hiroyuki
authors.identifier: 0000-0002-5041-5701
authors.initials: H
authors.lastname: Oyama
authors.affiliation: Kobe Pharmaceutical University , 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Izumi
authors.initials: I
authors.lastname: Morita
authors.affiliation: Kobe Pharmaceutical University , 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kiguchi
authors.affiliation: Kobe Pharmaceutical University , 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Erika
authors.initials: E
authors.lastname: Banzono
authors.affiliation: Kobe Pharmaceutical University , 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Kasumi
authors.initials: K
authors.lastname: Ishii
authors.affiliation: Kobe Pharmaceutical University , 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kubo
authors.affiliation: Kobe Pharmaceutical University , 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Yoshiro
authors.initials: Y
authors.lastname: Watanabe
authors.affiliation: Kobe Pharmaceutical University , 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Anna
authors.initials: A
authors.lastname: Hirai
authors.affiliation: Kobe Pharmaceutical University , 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Kaede
authors.affiliation: Kobe Pharmaceutical University , 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Ohta
authors.affiliation: Kobe Pharmaceutical University , 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Norihiro
authors.initials: N
authors.lastname: Kobayashi
mesh_terms: D000937:Antigen-Antibody Reactions; D002648:Child; D003367:Cotinine / Q000737:chemistry / Q000276:immunology / Q000652:urine*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008968:Molecular Conformation; D062105:Molecular Docking Simulation; D057127:Single-Chain Antibodies / Q000276:immunology
chemical_list: D057127:Single-Chain Antibodies; D003367:Cotinine