目的 建立Dickkopf-1(DKK-1)时间分辨免疫荧光分析(TR-IFMA)方法,并评价其在肺癌诊断和病理分期中的价值.方法 用双抗夹心原理建立DKK-1 TR-IFMA,并对其灵敏度、批内、批间差异和准确性等各项指标进行考核.同时用以测定212例肺癌、72例肺良性疾病患者和120名健康人血清DKK-1含量,以分析血清DKK-1水平与肺癌不同临床病理特性的相关性.结果 DKK-1TR-IFMA的稳定性好,线性宽,其检测灵敏度为0.08μg/L,批内和批间变异系数均<6.5%,与商业ELISA试剂盒相关性好(r=0.972,P=0.01).肺癌组血清DKK-1水平[31.93(79.47～18.03)μg/L]显著高于肺良性疾病组[15.25(18.41～11.49)μg/L]和正常对照组[13.90(16.91～11.02)μg/L],但DKK-1水平与年龄、性别、肺癌病理组织类型无关,与TNM分期(r=0.37,P<0.001)、有无淋巴结转移(r=0.52,P<0.001)和远处转移(r=0.62,P=0.001)显著相关;血清DKK-1诊断肺癌的总敏感度为68.4%,特异度为92.2%,准确性为82.1%,阳性预测值为90.6%,阴性预测值为72.5%;血清DKK-1诊断小细胞性肺癌的敏感度和准确性稍高于非小细胞性肺癌(70.7%vs 69.5%,85.6%vs 80.7%).结论 DKK-1可作为一项肺癌血清学标志,适用于肺癌的辅助诊断和病理分期.TR-IFMA检测血清DKK-1方法准确可靠,且为肺癌的DKK-1定量检测提供了技术平台.%Objective To develop a highly sensitive and accurate time-resolved immunofluorometric assay (TR-IFMA) for measurement of Dickkopf-1(DKK-1),as a novel serologic biomarker for lung cancer. Methods The study constructed a two-manoclonal-antibody "sandwich"-type assay and the sensitivity, within run CV and between run CV and accuracy were evaluated. Serum DKK-1 concentrations were measured by TR-IFMA in 120 healthy controls, in 72 benign lung disease patients, and in 212 lung cancer patients before surgery. The association between serum DKK-1 levels and clinicopathological features were evaluated. Results A standard curve for DKK-1 TR-IFMA had been developed with good sensitivity (0.08 μg/L). Both within run CV and between run CV were less than 6.5%. Accuracy studies, parallelism and precision data were determined and all found to be satisfactory. The validity of DKK-1 assay was confirmed by the good correlation between the results obtained by TR-IFMA and commercial ELISA (r=0.972, P=0.01). The serum levels of DKK-1 were higher in lung cancer patients 31.93(79.47-18.03) μg/L than in benign lung diseases 15.25(18.41-11.49) μg/L and in healthy controls 13. 90( 16. 91-11.02) μg/L DKK-1 levels were significant associated with the presence of distant metastases, as well as lymph node metastases and TNM stage, but not with patients' age, gender and tumor histology. At the cutoff of 22.63 μg/L, the diagnostic sensitivity, specificity, accuracy, positive predictive value and negative predictive value of the TR-IFMA for lung carcinoma were 68.4%, 92.2%, 82.1% ,90.6% and 72.5%. Diagnostic sensitivity and accuracy were higher for small cell carcinoma than for non-small cell carcinoma (70.7% vs 69.5% and 85.6% vs 80.7%, respectively). Conclusions A highly sensitive and reliable TR-IFMA for DKK-1 has been developed. The determination of serum DKK-1 levels may be useful for diagnosis and tumor staging of lung cancer.
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