Development and potential application of a simultaneous multiplex assay of Golgi protein 73 and alpha-fetoprotein for hepatocellular carcinoma diagnosis

J. Xiao, F. Long, T. Peng, L.-B. Hu, H. Cai, R. Chen, W.-L. Chen

State Key Laboratory of Respiratory Disease, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, Guangzhou, China. wenlichendoc@aliyun.com

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• Oncology

OBJECTIVE: Detecting a single serum marker, such as Golgi protein 73 (GP73) or alpha-fetoprotein (AFP), may not meet the requirements for the early diagnosis of hepatocellular carcinoma (HCC) due to low sensitivity and specificity. Therefore, this study aimed to develop a simultaneous multiplex assay of GP73 and AFP.
PATIENTS AND METHODS: Anti-human GP73- and AFP-coupled microsphere beads and biotin-labeled detectable antibodies were prepared to develop a multiplex assay of GP73 and AFP using the Luminex xMAP technology. The assay was evaluated for cross-reactivity, standard curve, sensitivity, range of detection, and precision. Additionally, the assay was used to determine the levels of serum GP73 and AFP in healthy controls and patients with chronic hepatitis, liver cirrhosis, and HCC.
RESULTS: The multiplex assay was successfully developed to simultaneously detect GP73 and AFP without cross-reactivity. The sensitivity for GP73 detection was 0.215 ng/mL and that for AFP detection was 0.666 ng/mL. The ranges of GP73 and AFP detection were 0.98-861.08 ng/mL and 2.01-1848.73 ng/mL, respectively. The intra- and inter-assay coefficients of variation (CVs) were <10%, indicating good precision, with recovery rates of 75-125%. The levels of serum GP73 in healthy controls, chronic hepatitis patients, liver cirrhosis patients, and HCC patients were 61.64 ± 30.60 ng/mL, 208.4 ± 99.42 ng/mL, 183.7 ± 82.78 ng/mL, and 214.1 ± 160.5 ng/mL, respectively. The levels of serum AFP in healthy controls, chronic hepatitis patients, liver cirrhosis patients, and HCC patients were 24.87 ± 14.52 ng/mL, 134.4 ± 216.5 ng/mL, 66.45 ± 133.4 ng/mL, and 891.4 ± 1278 ng/mL, respectively. The receiver operating characteristic (ROC) results showed that the area under the curves (AUC) for the combination of GP73 and AFP was 0.972, which was larger than the AUC for each marker. The sensitivity and specificity of the combined detection of GP73 and AFP for the diagnosis of HCC were 90.91% and 98.86%, respectively. The multiplex assay demonstrated a good correlation with enzyme-linked immunosorbent assay (ELISA), with correlation coefficients of 0.818 and 0.982 for GP73 (p<0.001) and AFP (p<0.001), respectively.
CONCLUSIONS: A multiplex assay for the simultaneous detection of GP73 and AFP with high sensitivity and accuracy was developed for the diagnosis of HCC. This assay may provide a reliable reference for the early diagnosis of HCC.

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