血清AFP-L3免疫放射分析与化学发光免疫分析试剂盒的制备及其临床应用研究

Preparation and clinical application study of serum AFP-L3 protein immunoradiometric assay and chemiluminescent immunoassay kits

    摘要
  • 摘要:
    目的  制备血清甲胎蛋白异质体L3（AFP-L3）免疫放射分析（IRMA）和化学发光免疫分析（CLIA）试剂盒，系统评估其分析性能，并探索这2种检测方法在肝细胞性肝癌（HCC）临床诊断中的应用价值。
    方法 将一株AFP-L3 单克隆抗体固定在羧基磁微粒表面作为固相载体，另一株单克隆抗体分别用 125I 放射性标记和吖啶酯化学发光标记，制备AFP-L3 IRMA和AFP-L3 CLIA试剂盒，对比分析其检测AFP-L3的灵敏度、精密度和回收率等关键性能指标。通过检测正常对照组（n=50）、肝硬化组（n=54）和HCC组（n=83）的血清样本，评价2种试剂盒的诊断效能。计量资料的2组间比较采用Mann-Whitney U检验，多组间比较采用Kruskal-Wallis H检验。采用受试者工作特征（ROC）曲线评估AFP-L3%（即AFP-L3占总AFP的比例）对HCC的诊断效能。
    结果  制备的AFP-L3 IRMA和AFP-L3 CLIA对AFP-L3的分析灵敏度分别为0.17 ng/ml和0.13 ng/ml，批内和批间变异系数均＜10%，样本回收率均值在90%~100%之间。2种试剂盒检测结果显示，肝硬化组和HCC组的AFP、AFP-L3水平均高于正常对照组，且差异均有统计学意义（Z=8.013~9.005，均P<0.05）；3组血清AFP-L3%间的差异均有统计学意义（H=120.563、120.732，均P<0.05）。ROC曲线分析结果显示，血清AFP诊断HCC的曲线下面积（AUC）为0.787（95%CI：0.722~0.852），灵敏度为0.690，特异度为0.780；血清AFP-L3%诊断HCC的AUC为0.840（95%CI：0.777~0.903），灵敏度为0.792，特异度为0.943。
    结论 制备的AFP-L3 IRMA和AFP-L3 CLIA试剂盒各项技术指标良好，可用于HCC的早期筛查以及临床辅助诊断。

     

    Abstract:
    Objective To prepare serum alpha-fetoprotein isoform L3 (AFP-L3) immunoradiometric assay (IRMA) and chemiluminescence immunoassay (CLIA) kits, systematically evaluate their analytical performance, and explore their application value in the clinical diagnosis of hepatocellular carcinoma (HCC).
    Methods One AFP-L3 monoclonal antibody was immobilized onto carboxyl-modified magnetic microparticles to serve as the solid-phase carrier, whereas another monoclonal antibody was labeled with 125I for radioisotope detection and with acridinium ester for chemiluminescence detection for the preparation of AFP-L3 IRMA and AFP-L3 CLIA kits. Key performance indicators, such as the sensitivity, precision, and recovery rate of AFP-L3 detection, were compared and analyzed. Serum samples from the normal control group (n=50), liver cirrhosis group (n=54), and HCC group (n=83) were detected to evaluate the diagnostic efficacy of the two kits. For measurement data that did not follow a normal distribution, Mann–Whitney U test was used for comparison between two groups, and Kruskal–Wallis H test was used for comparison among multiple groups. The diagnostic efficacy of AFP-L3% (i.e., the proportion of AFP-L3 in total AFP) for HCC was evaluated using the receiver operating characteristic (ROC) curve.
    Results The analytical sensitivity values of the prepared AFP-L3 IRMA and AFP-L3 CLIA for AFP-L3 were 0.17 and 0.13 ng/ml, respectively. The intra- and inter-assay variabilities were both less than 10%, and the mean recovery rate of samples was between 90% and 100%. The detection results of the two kits showed that the levels of AFP and AFP-L3 in the liver cirrhosis and HCC groups were higher than those in the normal control group, and the differences were statistically significant (Z=8.013–9.005, all P<0.05). The differences in serum AFP-L3% among the three groups were statistically significant (H=120.563, 120.732; both P<0.05). ROC curve analysis showed that the area under the curve (AUC) for the diagnosis of HCC by serum AFP was 0.787(95%CI: 0.722–0.852), with a sensitivity of 0.690 and a specificity of 0.780, the AUC for the diagnosis of HCC by serum AFP-L3% was 0.840 (95%CI: 0.777–0.903), with a sensitivity of 0.792 and a specificity of 0.943.
    Conclusion The prepared AFP-L3 IRMA and AFP-L3 CLIA kits have good technical indicators, and they can be used for the early screening and clinical auxiliary diagnosis of HCC.

     

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