An automated flow immunosensor based on kinetic exclusion analysis for measurement of a free β-subunit of human chorionic gonadotropin in serum

Abstract

An automated immunosensor assay has been developed and validated for measurement of the β-subunit of human chorionic gonadotropin (β-hCG). The assay employed the flow kinetic-exclusion analytical technology of the KinExA™ 3200 instrument. Our KinExA™ consisted of a capillary flow observation cell with a microporous screen that maintained a compact antigen-coated bead bed, polymethylmethacrylate (PMMA) beads coated with β-hCG. Various concentrations of β-hCG were incubated with a fixed amount of mouse anti-β-hCG monoclonal antibody until the binding reaction reached equilibrium. These solutions were then passed rapidly over the β-hCG-coated PMMA beads. Anti-β-hCG antibody not bound to the soluble β-hCG was captured by the β-hCG immobilized on the beads. The unbound reagents were removed from the bead bed by subsequent flushing with phosphate-buffered saline (PBS). The beads were then subjected to brief contact with a fluorescent-labeled secondary antibody, followed by a second PBS flush. The fluorescence was monitored and recorded during the flow of the fluorescent-labeled antibody through the bead bed. The fluorescence signal retained on the beads was replotted versus β-hCG concentrations to generate a calibration curve. The concentrations of β-hCG in the samples were then obtained by interpolation on the curve. The assay limit of detection was 0.2 ng mL⁻¹. This highly sensitive automated system allowed rapid and reliable quantification of β-hCG without any matrix effect; analytical recovery of serum-spiked β-hCG was 96.9–105.4 ± 3.54–6.84%. The precision of the sensor was satisfactory; the relative standard deviation (RSD) was 3.57–5.61 and 4.23–6.81% for the intra- and inter-assay precision, respectively. The analytical performance of the proposed sensor was found to be superior to the reported enzyme-linked immunosorbent assays for β-hCG. The automated analysis by the sensor facilitated the processing of a large number of specimens, and the new sensor-based assay is anticipated to have a great value in measurement of β-hCG.