B-357 Development of a highly sensitive Chemiluminescent Enzyme Immunoassay for Glypican-3

Background Hepatocellular carcinoma (HCC) is one of the most lethal causes of cancer-related death worldwide. There are often no symptoms of liver cancer until the later stages, which makes the treatment of HCC challenging and necessitates early detection. Serum biomarkers could be one of the options to detect early HCC, but the sensitivity is still limited. Glypican-3 (GPC3) is highly expressed in HCC and has become a reliable diagnostic marker for HCC by immunohistochemical studies. Therefore, detection of GPC3 in serum could be a biomarker for primary HCC, but it is still controversial. One of the reasons is that solid assay systems for GPC3 have not been established. Therefore, it is necessary to establish a rapid and simple method to measure GPC3. We developed a highly sensitive and quantitative chemiluminescence enzyme-immunoassay (CLEIA) for GPC3 on LUMIPULSE® platform. The aim of this study was to determine the preliminary performance of the newly developed GPC3 assay. Methods The reagent was developed and validated on a fully automated immunoassay system, LUMIPULSE L2400 (FUJIREBIO INC). Twenty-microliter specimens were assayed using a two-step sandwich immunoassay. The Limit of Detection (LoD), Limit of Quantification (LoQ), within-laboratory precision, linearity, interference, and cross reactivity with endogenous substances were determined. Furthermore, the clinical utility of the GPC3 assay in HCC detection was compared with existing cancer markers, AFP and PIVKA-II. Data were analyzed using R or Microsoft Excel statistical tool Analyse-it. Results The LoD and the LoQ were 1.7 pg/mL and 4.1 pg/mL, respectively. The within-laboratory precision showed CV 2.1-2.7 %. Linearity was demonstrated over the range 3.3-12154.0 pg/mL. No interference was observed with unconjugated (=20.4 mg/dL) or conjugated bilirubin (=20.4 mg/dL), chyle (=1560 FTU), RF (rheumatoid factor, =666 IU/mL), and HAMA (human anti-mouse antibody, =1249 ng/mL). Cross reactivity was confirmed to be less than 0.1% with Glypican family (1, 2, 4, 5, and 6). The AUCs of GPC3, AFP and PIVKA-II assays were 0.90, 0.89 and 0.76, respectively (N=180). Conclusion This preliminary performance evaluation of the GPC3 assay indicated promising results in terms of low variability, high sensitivity, and clinical utility. These data demonstrate that GPC3 assay will be useful in enhancing the detection of HCC. Moreover, since various inhibitors targeting GPC3 are currently under investigation, this LUMIPULSE GPC3 assay would be useful to screen the appropriate patient.