title: Rapid sandwich ELISA-based in vitro diagnostic procedure for the highly-sensitive detection of human fetuin A.
abstract: A rapid sandwich enzyme-linked immunosorbent assay (ELISA)-based in vitro diagnostic (IVD) procedure has been developed for human fetuin A (HFA), an important disease biomarker for inflammatory diseases as well as malignancies. In this simplified and cost-effective procedure, the EDC-activated anti-HFA antibody (Ab) was admixed with 1% (v/v) 3-aminopropyltriethoxysilane (APTES) in 1:1 (v/v) and dispensed in a KOH-pretreated microtiter plate (MTP). APTES formed a stable complex with the capture antibody that was in turn covalently bonded on the KOH-treated surface in 30 min. The resulting immunoassay (IA) format detects HFA with a dynamic range of 0.1-243 ng mL(-1), and a limit of detection (LOD) and analytical sensitivity of 0.3 ng mL(-1) and 1.0 ng mL(-1), respectively. For the determination of HFA spiked in diluted human whole blood and serum, and HFA in ethylenediaminetetraacetic acid (EDTA)-plasma of patients, the obtained analytical precision is similar to that of the conventional sandwich ELISA. The anti-HFA Ab-bound MTPs, stored at 4 °C in 0.1M PBS, pH 7.4, retained its biological activity for 8 weeks, thereby demonstrating excellent storage stability. This generic sandwich ELISA procedure can be extended for rapid, simplified and cost-effective detection of other disease biomarkers.
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: D015374:Biosensing Techniques; D001774:Blood Chemical Analysis; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D009369:Neoplasms / Q000097:blood / Q000175:diagnosis; D060749:alpha-2-HS-Glycoprotein / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D060749:alpha-2-HS-Glycoprotein
keywords: APTES; EDC; Human fetuin A; In vitro diagnostics; Rapid sandwich ELISA