Abstract
An automated immunosensor assay has been developed and validated for measurement of the beta-subunit of human chorionic gonadotropin (beta-hCG). The assay employed the flow kinetic-exclusion analytical technology of the KinExA (TM) 3200 instrument. Our KinExA (TM) consisted of a capillary flow observation cell with a microporous screen that maintained a compact antigen-coated bead bed, polymethylmethacrylate (PMMA) beads coated with beta-hCG. Various concentrations of beta-hCG were incubated with a fixed amount of mouse anti-beta-hCG monoclonal antibody until the binding reaction reached equilibrium. These solutions were then passed rapidly over the beta-hCG-coated PMMA beads. Anti-beta-hCG antibody not bound to the soluble beta-hCG was captured by the beta-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 beta-hCG concentrations to generate a calibration curve. The concentrations of beta-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 beta-hCG without any matrix effect; analytical recovery of serum-spiked beta-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 beta-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 beta-hCG.