Rapid sandwich ELISA-based in vitro diagnostic procedure for the highly-sensitive detection of human fetuin A.

Sandeep Kumar Vashist, E Marion Schneider, John H T Luong

Index: Biosens. Bioelectron. 67 , 73-8, (2015)

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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.Copyright © 2014 Elsevier B.V. All rights reserved.