肝癌标志物受体酪氨酸激酶AXL、软骨寡聚基质蛋白和骨桥蛋白多重检测试剂开发与验证

Development and validation of the multiplex immunoassay of serum markers for liver cancer:receptor tyrosine kinase AXL,cartilage oligomeric matrix protein and osteopontin

目的:基于流式荧光技术开发血清中肝癌标志物受体酪氨酸激酶AXL、软骨寡聚基质蛋白(COMP)和骨桥蛋白(OPN)的多重检测试剂。方法:建立检测血清甲胎蛋白(AFP)、AXL、COMP和OPN的流式荧光免疫法,并进行检测性能分析,筛选两个队列的肝癌血清样本进行临床验证并初步探讨四项标志物对肝癌的诊断价值。结果:AFP、AXL、COMP和OPN的检测范围依次为0.046~100.000、0.014~30.000、0.007~15.000、0.023~50.000 ng/mL,最低检测限依次为0.010、0.014、0.004、0.007 ng/mL,批内精密度<8%,批间精密度<5%,回收率为84%~101%,线性度为95%~102%。AXL、COMP和OPN之间没有观察到交叉反应。流式荧光免疫法与临床化学发光免疫法具有显著相关性(r=0.995,P<0.001)。肝癌血清AFP、AXL、COMP和OPN水平均高于健康对照,差异有统计学意义(P<0.01)。针对所有血清样本(n=100),绘制肝癌与健康对照的受试者工作特征(ROC)曲线,血清OPN的曲线下面积(AUC)为0.913,在单项标志物中,OPN对肝癌的诊断效果最佳;AFP、AXL和OPN组合的AUC为0.937,AFP、AXL、COMP和OPN组合的AUC为0.940。结论:本研究建立了四项肝癌血清标志物联合检测方法。AXL、COMP和OPN是诊断肝癌的潜在血清标志物,AXL、OPN与AFP联合检测的肝癌预测模型展示出较好的检测性能。结果对进一步开发其他标志物的多重检测试剂具有重要的参考价值。

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.