Application of microTAS (micro Total Analysis Systems) technologies utilizing chips with microfluidic channels to clinical diagnostic testing has drawn a lot of attention since it is expected to contribute to shortening reaction time, reduction of reagent/sample consumption, reducing instrument size, and other advantages of microchip electrophoresis. We have developed a fully automated immunoassay system by employing isotachophoresis followed by capillary gel electrophoresis for immunoreaction and B/F separation in microfluidic channels on polymer microchips. Laser-Induced Fluorescence (LIF) was used for detection of the sandwich immunocomplex composed of DNA-conjugate antibody, antigen and fluorescent dye-conjugated antibody. An immunoassay for PIVKA II was demonstrated on this new microTAS system utilizing the DNA-conjugated anti PIVKA II antibody and the fluorescent-dye labeled anti-prothrombin antibody. The resulting assay showed good assay performance with high sensitivity (LOD = 5mAU/mL), good reproducibility(CV = 1.0 - 5.7%) and good correlation with the commercially available PIVKA II assay kit (regression curve of y = 1.04x + 11.1, r = 0.991). The assay turn around time (TAT) was about 9 min. The PIVKA II assay will be useful for the diagnosis and prognosis of hepatocellular carcinoma.
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