One-Shot In Vitro Evolution Generated an Antibody Fragment for Testing Urinary Cotinine with More Than 40-Fold Enhanced Affinity
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 Ka value of 2.7 × 107 M-1 (at 4 °C) was prepared by linking the VH and VL 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 Ka (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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2017 |
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| Erschienen: |
2017 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:89 |
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| Enthalten in: |
Analytical chemistry - 89(2017), 1 vom: 03. Jan., Seite 988-995 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Oyama, Hiroyuki [VerfasserIn] |
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| Links: |
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| Themen: |
Cotinine |
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| Anmerkungen: |
Date Completed 05.11.2018 Date Revised 05.11.2018 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1021/acs.analchem.6b04332 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 245 | 1 | 0 | |a One-Shot In Vitro Evolution Generated an Antibody Fragment for Testing Urinary Cotinine with More Than 40-Fold Enhanced Affinity |
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| 520 | |a 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 Ka value of 2.7 × 107 M-1 (at 4 °C) was prepared by linking the VH and VL 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 Ka (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 | ||
| 650 | 4 | |a Journal Article | |
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| 700 | 1 | |a Morita, Izumi |e verfasserin |4 aut | |
| 700 | 1 | |a Kiguchi, Yuki |e verfasserin |4 aut | |
| 700 | 1 | |a Banzono, Erika |e verfasserin |4 aut | |
| 700 | 1 | |a Ishii, Kasumi |e verfasserin |4 aut | |
| 700 | 1 | |a Kubo, Satoshi |e verfasserin |4 aut | |
| 700 | 1 | |a Watanabe, Yoshiro |e verfasserin |4 aut | |
| 700 | 1 | |a Hirai, Anna |e verfasserin |4 aut | |
| 700 | 1 | |a Kaede, Chiaki |e verfasserin |4 aut | |
| 700 | 1 | |a Ohta, Mitsuhiro |e verfasserin |4 aut | |
| 700 | 1 | |a Kobayashi, Norihiro |e verfasserin |4 aut | |
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