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.
Recombinant gpc3
Manufactured by R&D Systems
Sourced in United States
Recombinant GPC3 is a laboratory product manufactured by R&D Systems. It is a recombinant human Glypican-3 protein.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using recombinant gpc3
1
Biomarker Quantification: AFP, PIVKA-II, and FL-GPC3
2
Generation of Anti-GPC3 Monoclonal Antibodies
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Recombinant GPC3 (R&D Systems, Minneapolis, MN, USA) was used an antigen. All animal protocols were approved by the Animal Care and Use Committee of the National Cancer Center, Japan, and the experiments were performed in accordance with the institutional guidelines. Eight-week-old BALB/c mice (Charles River, Japan) were immunized with an emulsion of Recombinant GPC3 (10 μg/50 μL) and 50 μL Titer Max Gold (Titer-Max USA Inc., Norcross, GA, USA), followed by repeated immunization with 10 µg of Recombinant GPC3 in phosphate-buffered saline (PBS). Spleen cells were isolated and fused with mouse myeloma cells (SP2/O-Ag14; RIKEN BRC, Ibaraki, Japan) using PEG1500 (Roche Diagnostics GmbH, Mannheim, Germany). Hybridomas producing GPC3 mAbs were selected by ELISA, and cloning was performed by the limited dilution method. Twelve anti-GPC3 mAbs were generated; of these, we used two anti-GPC3 mAbs (10A4 and 2E11). Specifically, 10A4 was used as a capture antibody, and 2E11, which was biotinylated using EZ-Link Sulfo-NHS-LC-Biotin reagent (Thermo Fisher Scientific, San Jose, CA, USA), was used as a detection antibody.
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