title: One-step antibody immobilization-based rapid and highly-sensitive sandwich ELISA procedure for potential in vitro diagnostics.
abstract: An improved enzyme-linked immunosorbent (ELISA) assay using one-step antibody immobilization has been developed for the detection of human fetuin A (HFA), a specific biomarker for atherosclerosis and hepatocellular carcinoma. The anti-HFA formed a stable complex with 3-aminopropyltriethoxysilane (APTES) by ionic and hydrophobic interactions. The complex adsorbed on microtiter plates exhibited a detection range of 4.9 pg mL(-1) to 20 ng mL(-1) HFA, with a limit of detection of 7 pg mL(-1). Furthermore, an analytical sensitivity of 10 pg mL(-1) was achieved, representing a 51-fold increase in sensitivity over the commercial sandwich ELISA kit. The results obtained for HFA spiked in diluted human whole blood and plasma showed the same precision as the commercial kit. When stored at 4°C in 0.1 M phosphate-buffered saline (PBS, pH 7.4), the anti-HFA bound microtiter plates displayed no significant decrease in their functional activity after two months. The new ELISA procedure was extended for the detection of C-reactive protein, human albumin and human lipocalin-2 with excellent analytical performance.
authors.affiliation: 1] HSG-IMIT - Institut für Mikro- und Informationstechnik, Georges-Koehler-Allee 103, 79110 Freiburg, Germany [2] Laboratory for MEMS Applications, Department of Microsystems Engineering -IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
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
authors.initials: E
authors.lastname: Marion Schneider
authors.affiliation: National Research Council Canada, Montreal, Quebec, Canada H4P 2R2.
authors.forename: Edmond
authors.initials: E
authors.lastname: Lam
authors.affiliation: National Research Council Canada, Montreal, Quebec, Canada H4P 2R2.
authors.forename: Sabahudin
authors.initials: S
authors.lastname: Hrapovic
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: D000209:Acute-Phase Proteins; D055910:Antibodies, Immobilized / Q000737:chemistry; D000936:Antigen-Antibody Complex / Q000737:chemistry; D050197:Atherosclerosis / Q000097:blood / Q000175:diagnosis*; D015415:Biomarkers / Q000097:blood; D002097:C-Reactive Protein / Q000032:analysis; D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000175:diagnosis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D057230:Limit of Detection; D000071068:Lipocalin-2; D054834:Lipocalins / Q000097:blood; D008113:Liver Neoplasms / Q000097:blood / Q000175:diagnosis*; D011437:Propylamines; D011518:Proto-Oncogene Proteins / Q000097:blood; D012709:Serum Albumin / Q000032:analysis; D012821:Silanes / Q000737:chemistry; D060749:alpha-2-HS-Glycoprotein / Q000032:analysis*
chemical_list: D000209:Acute-Phase Proteins; D055910:Antibodies, Immobilized; D000936:Antigen-Antibody Complex; D015415:Biomarkers; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D011437:Propylamines; D011518:Proto-Oncogene Proteins; D012709:Serum Albumin; D012821:Silanes; D060749:alpha-2-HS-Glycoprotein; D002097:C-Reactive Protein; C477625:amino-propyl-triethoxysilane