Fully automated assay kit

Plasma GPC3 concentrations were measured using a fully automated assay kit provided by Sysmex Corporation (Kobe, Japan). Briefly, a biotinylated monoclonal antibody reagent was used to capture GPC3 from clinical plasma samples of patients. Further, streptavidin-coated magnetic beads were used to capture the immune complexes. Following magnetic separation and washing for B/F (bound/free fraction) separation, a second monoclonal antibody labeled with alkaline phosphatase was reacted with the immune complex. After a second round of B/F separation, the immune complex was quantified using the HISCL chemiluminescent reagent. All reactions were performed at 42°C in the HISCL–800 fully automated immunoassay system, within 17 minutes. Serum AFP and PIVKA-II concentrations in patients were measured at the time of plasma collection for detection of GPC3 using a commercially available electrochemiluminescence immunoassay kit (Roche Co., Tokyo, Japan) and a chemiluminescent enzyme immunoassay kit (Eisai Co., Tokyo, Japan), respectively. In this study, the cut-off values for AFP and PIVKA-II were set as 10 ng/mL and 40 mAU/mL, respectively.

Plasma GPC3 concentrations were measured using a fully automated assay kit provided by Sysmex Corporation (Kobe, Japan). Briefly, a biotinylated monoclonal antibody reagent was employed to capture GPC3 from clinical plasma samples from patients, and streptavidin-coated magnetic beads were used to capture the immune complexes. Following magnetic separation and washing for bound/free fraction (B/F) separation, a second monoclonal antibody labeled with alkaline phosphatase was reacted with the immune complex. After a second round of B/F separation, the immune complex was quantified using the HISCL chemiluminescent reagent (Sysmex Corporation). All reactions were performed at 42°C in the HISCL-800 fully automated immunoassay system (Sysmex Corporation) within 17 min.