Framework-Directed Amino-Acid Insertions Generated over 55-Fold Affinity-Matured Antibody Fragments That Enabled Sensitive Luminescent Immunoassays of Cortisol
We generated three single-chain Fv fragments (scFvs) specific to cortisol according to our original affinity-maturation strategy and verified their utility in developing immunoassays. These scFv mutants (m-scFvs) had insertion of one, four, or six amino acid(s) in the framework region 1 of the VH-do...
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| Veröffentlicht in: | Biological & pharmaceutical bulletin 2023/12/01, Vol.46(12), pp.1661-1665 |
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| creator | Kiguchi, Yuki Morita, Izumi Yamaki, Kouya Takegami, Shigehiko Kobayashi, Norihiro |
| description | We generated three single-chain Fv fragments (scFvs) specific to cortisol according to our original affinity-maturation strategy and verified their utility in developing immunoassays. These scFv mutants (m-scFvs) had insertion of one, four, or six amino acid(s) in the framework region 1 of the VH-domain and showed >55-fold higher affinity (Ka, 2.0 − 2.2 × 1010 M−1) than the unmodified scFv (wt-scFv). Each m-scFv was fused with NanoLuc luciferase (NLuc) for the use in enzyme-linked immunosorbent assays (ELISAs). In these ELISA, the m-scFv–NLuc fusions were competitively reacted with immobilized cortisol residues and cortisol standards, and then the bound NLuc activity was monitored luminometrically. The luminescent ELISAs generated dose–response curves with extremely low midpoints (approx. 3 pg/assay) and were >150-fold more sensitive than the colorimetric ELISAs using wt-scFv and >8000-fold more sensitive than the ELISA using the parental native antibody. The luminescent ELISAs showed acceptable cross-reactivity patterns with related steroids, and the determination of control sera afforded cortisol levels in the reference range with satisfactory parallelism. |
| doi_str_mv | 10.1248/bpb.b23-00656 |
| format | Article |
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These scFv mutants (m-scFvs) had insertion of one, four, or six amino acid(s) in the framework region 1 of the VH-domain and showed >55-fold higher affinity (Ka, 2.0 − 2.2 × 1010 M−1) than the unmodified scFv (wt-scFv). Each m-scFv was fused with NanoLuc luciferase (NLuc) for the use in enzyme-linked immunosorbent assays (ELISAs). In these ELISA, the m-scFv–NLuc fusions were competitively reacted with immobilized cortisol residues and cortisol standards, and then the bound NLuc activity was monitored luminometrically. The luminescent ELISAs generated dose–response curves with extremely low midpoints (approx. 3 pg/assay) and were >150-fold more sensitive than the colorimetric ELISAs using wt-scFv and >8000-fold more sensitive than the ELISA using the parental native antibody. The luminescent ELISAs showed acceptable cross-reactivity patterns with related steroids, and the determination of control sera afforded cortisol levels in the reference range with satisfactory parallelism.</description><identifier>ISSN: 0918-6158</identifier><identifier>EISSN: 1347-5215</identifier><identifier>DOI: 10.1248/bpb.b23-00656</identifier><language>eng</language><publisher>Tokyo: The Pharmaceutical Society of Japan</publisher><subject>Affinity ; affinity maturation ; Amino acids ; Colorimetry ; Cortisol ; Cross-reactivity ; Enzyme-linked immunosorbent assay ; framework region ; Hormones ; Immunoassay ; nanoluc luciferase ; single-chain Fv fragment (scFv) ; Steroid hormones</subject><ispartof>Biological and Pharmaceutical Bulletin, 2023/12/01, Vol.46(12), pp.1661-1665</ispartof><rights>2023 The Pharmaceutical Society of Japan</rights><rights>Copyright Japan Science and Technology Agency 2023</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c420t-dd785372c6a81fde8ab1284e1d76703a15836fe49dffaad2a0728bff0901a20f3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,1877,27901,27902</link.rule.ids></links><search><creatorcontrib>Kiguchi, Yuki</creatorcontrib><creatorcontrib>Morita, Izumi</creatorcontrib><creatorcontrib>Yamaki, Kouya</creatorcontrib><creatorcontrib>Takegami, Shigehiko</creatorcontrib><creatorcontrib>Kobayashi, Norihiro</creatorcontrib><title>Framework-Directed Amino-Acid Insertions Generated over 55-Fold Affinity-Matured Antibody Fragments That Enabled Sensitive Luminescent Immunoassays of Cortisol</title><title>Biological & pharmaceutical bulletin</title><description>We generated three single-chain Fv fragments (scFvs) specific to cortisol according to our original affinity-maturation strategy and verified their utility in developing immunoassays. These scFv mutants (m-scFvs) had insertion of one, four, or six amino acid(s) in the framework region 1 of the VH-domain and showed >55-fold higher affinity (Ka, 2.0 − 2.2 × 1010 M−1) than the unmodified scFv (wt-scFv). Each m-scFv was fused with NanoLuc luciferase (NLuc) for the use in enzyme-linked immunosorbent assays (ELISAs). In these ELISA, the m-scFv–NLuc fusions were competitively reacted with immobilized cortisol residues and cortisol standards, and then the bound NLuc activity was monitored luminometrically. The luminescent ELISAs generated dose–response curves with extremely low midpoints (approx. 3 pg/assay) and were >150-fold more sensitive than the colorimetric ELISAs using wt-scFv and >8000-fold more sensitive than the ELISA using the parental native antibody. The luminescent ELISAs showed acceptable cross-reactivity patterns with related steroids, and the determination of control sera afforded cortisol levels in the reference range with satisfactory parallelism.</description><subject>Affinity</subject><subject>affinity maturation</subject><subject>Amino acids</subject><subject>Colorimetry</subject><subject>Cortisol</subject><subject>Cross-reactivity</subject><subject>Enzyme-linked immunosorbent assay</subject><subject>framework region</subject><subject>Hormones</subject><subject>Immunoassay</subject><subject>nanoluc luciferase</subject><subject>single-chain Fv fragment (scFv)</subject><subject>Steroid hormones</subject><issn>0918-6158</issn><issn>1347-5215</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2023</creationdate><recordtype>article</recordtype><recordid>eNpdkT1v2zAQhoWgBeKmGbMT6NKFKT_0QY2GE6cGXHRoOhMn6ZjQlUiXpBL41_Svlo4LD-VADu_z3h3vLYobzm65KNWXbt_ddkJSxuqqvigWXJYNrQSv3hUL1nJFa16py-JDjDvGWMOEXBR_1gEmfPXhF72zAfuEA1lO1nm67O1ANi5iSNa7SB7QYYCj7l8wkKqiaz9m2BjrbDrQb5DmcHS7ZDs_HEiu_DShS5E8PkMi9w66Mes_0EWb7AuS7ZwbYewzQzbTNDsPMcIhEm_Iyue20Y8fi_cGxojX_96r4uf6_nH1lW6_P2xWyy3tS8ESHYZGVbIRfQ2KmwEVdFyoEvnQ1A2TkD8ua4NlOxgDMAhgjVCdMaxlHAQz8qr4fKq7D_73jDHpyebJxhEc-jlqodqmVPk0Gf30H7rzc3B5Oi1aXsq8ackzRU9UH3yMAY3eBztBOGjO9DEunePSOS79Flfm7078LiZ4wjMNeQ_9iG90WWfn8T7bznL_DEGjk38BvU2jWQ</recordid><startdate>20231201</startdate><enddate>20231201</enddate><creator>Kiguchi, Yuki</creator><creator>Morita, Izumi</creator><creator>Yamaki, Kouya</creator><creator>Takegami, Shigehiko</creator><creator>Kobayashi, Norihiro</creator><general>The Pharmaceutical Society of Japan</general><general>Japan Science and Technology Agency</general><scope>AAYXX</scope><scope>CITATION</scope><scope>7QP</scope><scope>7QR</scope><scope>7TK</scope><scope>7U9</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20231201</creationdate><title>Framework-Directed Amino-Acid Insertions Generated over 55-Fold Affinity-Matured Antibody Fragments That Enabled Sensitive Luminescent Immunoassays of Cortisol</title><author>Kiguchi, Yuki ; Morita, Izumi ; Yamaki, Kouya ; Takegami, Shigehiko ; Kobayashi, Norihiro</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c420t-dd785372c6a81fde8ab1284e1d76703a15836fe49dffaad2a0728bff0901a20f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2023</creationdate><topic>Affinity</topic><topic>affinity maturation</topic><topic>Amino acids</topic><topic>Colorimetry</topic><topic>Cortisol</topic><topic>Cross-reactivity</topic><topic>Enzyme-linked immunosorbent assay</topic><topic>framework region</topic><topic>Hormones</topic><topic>Immunoassay</topic><topic>nanoluc luciferase</topic><topic>single-chain Fv fragment (scFv)</topic><topic>Steroid hormones</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kiguchi, Yuki</creatorcontrib><creatorcontrib>Morita, Izumi</creatorcontrib><creatorcontrib>Yamaki, Kouya</creatorcontrib><creatorcontrib>Takegami, Shigehiko</creatorcontrib><creatorcontrib>Kobayashi, Norihiro</creatorcontrib><collection>CrossRef</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Biological & pharmaceutical bulletin</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Kiguchi, Yuki</au><au>Morita, Izumi</au><au>Yamaki, Kouya</au><au>Takegami, Shigehiko</au><au>Kobayashi, Norihiro</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Framework-Directed Amino-Acid Insertions Generated over 55-Fold Affinity-Matured Antibody Fragments That Enabled Sensitive Luminescent Immunoassays of Cortisol</atitle><jtitle>Biological & pharmaceutical bulletin</jtitle><date>2023-12-01</date><risdate>2023</risdate><volume>46</volume><issue>12</issue><spage>1661</spage><epage>1665</epage><pages>1661-1665</pages><artnum>b23-00656</artnum><issn>0918-6158</issn><eissn>1347-5215</eissn><abstract>We generated three single-chain Fv fragments (scFvs) specific to cortisol according to our original affinity-maturation strategy and verified their utility in developing immunoassays. These scFv mutants (m-scFvs) had insertion of one, four, or six amino acid(s) in the framework region 1 of the VH-domain and showed >55-fold higher affinity (Ka, 2.0 − 2.2 × 1010 M−1) than the unmodified scFv (wt-scFv). Each m-scFv was fused with NanoLuc luciferase (NLuc) for the use in enzyme-linked immunosorbent assays (ELISAs). In these ELISA, the m-scFv–NLuc fusions were competitively reacted with immobilized cortisol residues and cortisol standards, and then the bound NLuc activity was monitored luminometrically. The luminescent ELISAs generated dose–response curves with extremely low midpoints (approx. 3 pg/assay) and were >150-fold more sensitive than the colorimetric ELISAs using wt-scFv and >8000-fold more sensitive than the ELISA using the parental native antibody. The luminescent ELISAs showed acceptable cross-reactivity patterns with related steroids, and the determination of control sera afforded cortisol levels in the reference range with satisfactory parallelism.</abstract><cop>Tokyo</cop><pub>The Pharmaceutical Society of Japan</pub><doi>10.1248/bpb.b23-00656</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| source | J-STAGE (Japan Science & Technology Information Aggregator, Electronic) Freely Available Titles - Japanese; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Free Full-Text Journals in Chemistry |
| subjects | Affinity affinity maturation Amino acids Colorimetry Cortisol Cross-reactivity Enzyme-linked immunosorbent assay framework region Hormones Immunoassay nanoluc luciferase single-chain Fv fragment (scFv) Steroid hormones |
| title | Framework-Directed Amino-Acid Insertions Generated over 55-Fold Affinity-Matured Antibody Fragments That Enabled Sensitive Luminescent Immunoassays of Cortisol |
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