Abstract
An automated flow immunosensor has been developed and validated for the measurement of the cancer marker alpha-fetoprotein (AFP) in serum. The sensor employed the kinetic-exclusion analysis using the KinExA (TM) 3200 instrument. Various concentrations of AFP were incubated with a fixed amount of mouse anti-AFP monoclonal antibody until the binding reaction reached equilibrium. These solutions were then passed rapidly over AFP coated onto polymethylmethacrylate beads contained in the observation cell of the KinExA instrument. The anti-AFP antibody captured by the beads was quantified by passing a fluorescent-labeled secondary antibody over the beads, and the fluorescence signals were monitored during its flow. Calibration curve was generated by plotting the fluorescence signals that were retained on the beads as a function of AFP concentrations. The assay limit of detection was 8 ng/ml, and the working range of the assay was 8-500 ng/ml. Analytical recovery of serum-spiked AFP was 93.8-105.8 +/- 2.8-8.1%. The precision of the sensor was satisfactory; RSD was <= 7.2%. The proposed sensor was found to be superior to the existing conventional enzyme-linked immunosorbent assay (ELISA) for AFP. The sensor is anticipated to have a great value in measurement of AFP when a more confident result is needed.