title: A rapid sandwich immunoassay for human fetuin A using agarose-3-aminopropyltriethoxysilane modified microtiter plate.
abstract: A rapid sandwich immunoassay (IA) with enhanced signal response for human fetuin A (HFA) was developed by modifying the surface of a KOH-treated polystyrene microtiter plate (MTP) with agarose and 3-aminopropyltriethoxysilane (APTES). The agarose-APTES complex binds covalently to the hydroxyl moiety of the MTP plate to serve as a binding platform for bioconjugation of EDC-activated anti-HFA antibody (Ab) via carbodiimide coupling. The one-step kinetics-based sandwich enzyme-linked immunosorbent assay (ELISA) enabled the detection of HFA in 30 min with a limit of detection (LOD) and a linear range of 0.02 ng mL(-1) and 1-243 ng mL(-1), respectively. It detected HFA spiked in diluted human whole blood and serum, and HFA in ethylenediaminetetraacetic acid (EDTA)-plasma of patients with high precision similar to that of conventional ELISA. The anti-HFA Ab-bound agarose-functionalized MTPs retained their functional activity after 6 weeks of storage in 0.1 M PBS, pH 7.4 at 4 °C.
authors.affiliation: Laboratory for MEMS Applications, Department of Microsystems Engineering-IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany; HSG-IMIT-Institut für Mikro- und Informationstechnik, Georges-Koehler-Allee 103, 79110 Freiburg, Germany. Electronic address: sandeep.kumar.vashist@hsg-imit.de.
authors.forename: Sandeep Kumar
authors.initials: SK
authors.lastname: Vashist
authors.affiliation: Sektion Experimentelle Anaesthesiologie, University Hospital Ulm, Albert Einstein Allee 23, 89081 Ulm, Germany.
authors.forename: E Marion
authors.initials: EM
authors.lastname: Schneider
authors.affiliation: Innovative Chromatography Group, Irish Separation Science Cluster (ISSC), Department of Chemistry and Analytical, Biological Chemistry Research Facility (ABCRF), University College Cork, Cork, Ireland.
authors.forename: John H T
authors.initials: JH
authors.lastname: Luong
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry*; D006801:Humans; D007118:Immunoassay / Q000191:economics / Q000379:methods*; D057230:Limit of Detection; D011437:Propylamines / Q000737:chemistry*; D012685:Sepharose / Q000031:analogs & derivatives*; D012821:Silanes / Q000737:chemistry*; D060749:alpha-2-HS-Glycoprotein / Q000032:analysis*
chemical_list: D055910:Antibodies, Immobilized; D011437:Propylamines; D012821:Silanes; D060749:alpha-2-HS-Glycoprotein; D012685:Sepharose; C477625:amino-propyl-triethoxysilane
keywords: Agarose; Human fetuin A; One-step kinetics; Rapid sandwich immunoassay; Signal enhancement