AFP电化学发光免疫分析方法的建立

目的建立一种快速、特异、灵敏的检测人甲胎蛋白（AFP）的电化学发光免疫分析法。方法用链霉亲和素包被的磁性微粒、生物素标记的抗AFP单克隆抗体、钌复合物标记配对的抗AFP单克隆抗体组成AFP电化学发光免疫分析法试剂,在电化学发光免疫分析仪上对其准确性、灵敏度、特异性等效能进行方法学评价,同时用所建方法与进口的同类电化学发光免疫分析法试剂（Roche）对80例肝癌患者血清AFP检测结果进行相关性分析。对218名健康志愿者血清AFP进行了正常值调查。结果自建电化学发光免疫分析法检测AFP的批内精密度在2.8%～4.5%之间,批间精密度在3.2%～9.8%之间,分析灵敏度为0.605ng/ml。与进口同类试剂比较,直线回归方程为y=0.9936x-0.4566（r=0.9877,P〈0.05）。分析测量范围为0.605～1452.0ng/ml,自建试剂与CEA和CA199无交叉反应。自建电化学发光免疫分析法检测AFP的正常参考值为〈6.7ng/ml。结论自建AFP电化学发光免疫分析法特异性高,灵敏度好,与进口同类试剂检测结果相关性达0.9877。确定的实验室正常参考范围接近进口同类试剂,具备产业化的潜能。

Establishment of an electrochemiluminescence immunoassay for the detection of serum AFP

Objective To establish an electrochemiluminescence immunoassay for the detection of alpha-fetal protein（AFP） in human serum.Methods Homemade AFP kit containing biotinylated monoclonal AFP specific antibody,ruthenium labeled monoclonal AFP specific antibody,and streptavidin-coated microparticles was used in this study.The precision,sensitivity and specificity were evaluated.The results were compared with commercially available electrochemiluminescence immunoassay kit（Roche）.Clinical samples from 80 patients with primary hepatocellular carcinoma and 218 healthy volunteers were used for this testing.Results The precision was in the range of 2.8% to 4.5%,and the intermediate precision was 3.2%-9.8%.The detection limit was 0.605 ng/ml.The linear regression between the commercial and the homemade kit was y=0.9936x-0.4566（r=0.9877,P0.05）.The range of measurement was between 0.605 ng/ml and 1452.0 ng/ml.The homemade reagents were not cross-react with CEA and CA199.The expected value was lower than 6.7 ng/ml.Conclusion The homemade AFP electrochemiluminescence immunoassay kit is sensitive and specific for AFP.The accuracy in measurement is correlated with the commercial kit with a correlation index of 0.9877.The kit may further be developed for commercial application.