糖类抗原50化学发光免疫分析方法的建立及评价

Chemiluminescence Immunoassay for Quantitative Determinationof Carbohydrate Antigen 50 in Human Serum

目的建立以异硫氰酸荧光素(FITC)单抗包被磁微粒、吖啶酯为发光标记物的检测糖类抗原50(CA50)的磁微粒全自动化学发光免疫分析方法。方法根据双抗体夹心法原理,选择抗-FITC单克隆抗体偶联磁微粒,FITC标记一株抗-CA50单克隆抗体,吖啶酯标记另一株抗CA50单克隆抗体,建立CA50化学发光免疫分析方法,同时对该方法的空白限、检出限、线性范围、准确度、重复性、干扰进行了验证,并与迈瑞全自动化学发光试剂检测结果进行了比对。结果本方法最终采用两步法的反应模式,空白限为0.03U/mL,检出限为2U/mL,线性范围为4.0~500.0U/mL,样本的回收率为99.39%,高、低两份样本变异系数分别为4.05%和2.16%,与迈瑞全自动化学发光免疫测定系统同时测定131份样本,回归方程为Y=1.0177 X-3.0827,r=0.993。同时发现样本中的荧光素钠(FITC-Na)高于2μg/mL时会对本方法造成干扰。结论成功建立了糖类抗原50的吖啶酯全自动化学发光免疫分析方法,各项指标均满足临床检测的要求,具有一定的临床应用价值。

Objective To establish a magnetic particle chemiluminescence method using FITC monoclonal antibody coated magnetic particles and acridinium ester as luminescent marker for determining carbohydrate antigen 50(CA50).Methods According to the principle of double antibody sandwich method,an anti-FITC monoclonal antibody was coated to magnetic particles,one anti-CA50 monoclonal antibody was labeled with small molecule FITC,and another anti-CA50 monoclonal antibody was labeled with acridinium ester.At the same time,the chemiluminescence immunoassay system was established and evaluated by using a performance matrix,including LoQ,LOD,linear range,accuracy,repeatability and interference.Results With a two-step sampling mode,the limit of detection was 0.03 U/mL.The LOD was 2.0 U/mL.The measurement range of this method was 4.0 to 500.0 U/mL.The recovery rate of the samples was 99.93%.The Within-laboratory CV was 4.05%and 2.16%.And compared with Mindray CLIA system,the coefficient correlation was 0.990.Also,we discovered that when fluorescein sodium(FITC-Na)was higher than 2μg/mL,it would cause an interference to the study.Conclusion An acridinium aster-chemiluminescence of magnetic particles system for detecting CA50 has been established from the current study,which shows excellent performance indicators and can be widely used in clinical testing.