Chemiluminescent enzyme immunoassay kit

AFP and PIVKA-II were measured using an electrochemiluminescence immunoassay kit (Roche Co.) and chemiluminescent enzyme immunoassay kit (Eisai Co.), respectively. The assay system for FL-GPC3 was constructed using a sandwich immunoassay, in which a monoclonal mouse antibody against its N-terminus is used to capture the protein and a monoclonal mouse antibody against the C-terminus is used to detect the protein. The monoclonal antibody against its N-terminus was labeled with biotin and the antibody against its C-terminus with alkaline phosphatase. The immunoassay was performed by first reacting the plasma sample with the biotinylated antibody, then capturing the immunocomplex using streptavidin-coated magnetic beads. After washing the beads, an alkaline phosphatase-labeled antibody was added to form a sandwich immunocomplex. After a second wash, a luminescent substrate was added and the luminescence intensity was measured. All immunoassay steps were performed using a HISCL™ series (Sysmex Co.), which is an automated immunoassay device. Recombinant GPC3 (R&D Systems Inc.) was used as the assay standard. Standards at concentrations of 2, 15, 50, 150, 500, and 1,500 pg/ml were measured and calibration curves were generated by the four-parameter logistic regression method.

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.