title: Gaussia Luciferase as a Genetic Fusion Partner with Antibody Fragments for Sensitive Immunoassay Monitoring of Clinical Biomarkers.
abstract: In this study, we show the utility of Gaussia luciferase (GLuc), which is much smaller than previously found luciferases, as the fusion partner with artificial antibody species for developing sensitive immunoassay systems. As an example, we constructed a bioluminescent enzyme-linked immunosorbent assay (BL-ELISA) system determining the major glucocorticoid cortisol. A monoclonal antibody was newly elicited against a cortisol-albumin conjugate, and the genes encoding its variable domains (VH and VL) were cloned and combined to encode a single-chain Fv fragment (scFv). scFv was then linked to the wild-type GLuc gene or that encoding GLuc mutants reported to show improved emission kinetics and expressed in the periplasmic space of several Escherichia coli strains. Notably, the wild-type GLuc fusion protein (scFv-wtGLuc) showed the most suitable luminescent properties for BL-ELISAs. In our system, scFv-wtGLuc was reacted competitively with the analyte and immobilized cortisol moieties, and the bound GLuc activity was monitored with coelenterazine as the substrate. Successful batch-type luminescence detection was achieved using a plate reader without built-in injectors. The midpoint and limit of detection in a typical dose-response curve were 4.1 and 0.26 pg/assay, respectively, thus exhibiting much more sensitivity than conventional cortisol immunoassays. Serum cortisol levels (as the sum with cortisone) for healthy subjects, determined without any pretreatment, were compatible with reported reference ranges. The scFv-wtGLuc probe was stable over a year under storage as periplasmic extracts at -30 °C or with repeated freeze-thawing. These results suggest that GLuc fusions with antibody fragments might serve as useful and highly sensitive immunoassay probes in various clinical settings.
authors.affiliation: Kobe Pharmaceutical University , 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Hiroyuki
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: Sayaka
authors.initials: S
authors.lastname: Miyake
authors.affiliation: Kobe Pharmaceutical University , 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Moriuchi
authors.affiliation: Kobe Pharmaceutical University , 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Akisada
authors.affiliation: Department of Medical Technology, School of Health Sciences, Tohoku University , 2-1, Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Niwa
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: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D015415:Biomarkers / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006854:Hydrocortisone / Q000032:analysis*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D008156:Luciferases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D008163:Luminescent Measurements; D051379:Mice; D008807:Mice, Inbred BALB C; D015394:Molecular Structure; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D015415:Biomarkers; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D006854:Hydrocortisone