One-Shot In Vitro Evolution Generated an Antibody Fragment for Testing Urinary Cotinine with More Than 40-Fold Enhanced Affinity

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• 作者：Hiroyuki OyamaIzumi Morita ; Yuki Kiguchi ; Erika Banzono ; Kasumi Ishii ; Satoshi Kubo ; Yoshiro Watanabe ; Anna Hirai ; Chiaki Kaede ; Mitsuhiro Ohta ; Norihiro Kobayashi
• 刊名：Analytical Chemistry
• 出版年：2017
• 出版时间：January 3, 2017
• 年：2017
• 卷：89
• 期：1
• 页码：988-995
• 全文大小：469K
• ISSN：1520-6882

文摘

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 K_a value of 2.7 × 10⁷ M^–1 (at 4 °C) was prepared by linking the V_H and V_L domains 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 K_a (1.2 × 10⁹ M^–1 at 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.