Development and validation of the multiplex immunoassay of serum markers for liver cancer: receptor tyrosine kinase AXL, cartilage oligomeric matrix protein and osteopontin
LI Siping1, 2, HAN Jinyu3, HU Di4, WANG Yajie3, YU Xiaobo1, 2.
1.The First School of Medicine, School of Infomation and Engineering,Wenzhou Medical University, Wenzhou 325035, China; 2.National Center for Protein Science-Beijing (Phoenix Center), Beijing Proteomics Research Center, Beijing 102206, China; 3.Department of Laboratory, Beijing Ditan Hospital Affiliated to Capital Medical University, Beijing 100015, China; 4.ProteomicsEra Medical Ltd., Beijing 102206, China
LI Siping,HAN Jinyu,HU Di, et al. Development and validation of the multiplex immunoassay of serum markers for liver cancer: receptor tyrosine kinase AXL, cartilage oligomeric matrix protein and osteopontin[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2023, 53(9): 689-705,713.
Abstract：Objective: To develop a multiplex immunoassay of serum receptor tyrosine kinase AXL,cartilage oligomeric matrix protein (COMP) and osteopontin (OPN) for liver cancer detection. Methods: We developed a multiplex immunoassays containing alpha-fetoprotein (AFP), AXL, COMP and OPN using flow cytometry, which was tested on their performance in detecting liver cancer patients from two clinical cohorts.Results: The detection range for AFP, AXL, COMP and OPN was respectively 0.046-100.000, 0.014-30.000,0.007-15.000 and 0.023-50.000 ng/mL while the 1imit of detection was 0.010 ng/mL for AFP, 0.014 ng/mL for AXL, 0.004 ng/mL for COMP and 0.007 ng/mL for OPN. The intra-assay and inter-assay were below 8% and 5%, respectively. The recovery rate was 84%-101%, and the linearity was 95%-102%. The measurement for AXL, COMP and OPN showed that there was no significant cross-talking reaction. There was a high correlation between flow immunoassay and clinical chemiluminescence immunoassay (r=0.995, P<0.001). The level of AFP,AXL, COMP and OPN in liver cancer was all higher than that in healthy control, and the difference was statistically significant (P<0.01). In all serum samples (n=100), when receiver operating characteristic (ROC) curve was plotted for liver cancer versus healthy control, among the single markers, serum OPN with AUC 0.913 had the best diagnosis performance for liver cancer. The combination of AFP, AXL, OPN with area under the curve (AUC) 0.937 and the combination of four markers with AUC 0.940 had comparable diagnosis performance. Finally,AFP, AXL and OPN were used to generate liver cancer prediction model. Conclusion: We have successfully developed the assays for detecting four serum markers in HCC. AXL, COMP and OPN are potent serum markers for detection of liver cancer, and the liver cancer prediction model based on the combined of AXL, OPN and AFP showed excellent diagnostic performance. The results have important reference value for the further development of multiplex assay reagents for other markers.
