Objective To establish the time-resolved fluoroimmunoassay(TRFIA)of CA199 based on Mag-netic Microspheres(Nano-TRFIA).Methods Based on a sandwich-type immunoassay format,analytes in sam-ples were captured by magnetic particles coated with anti-CA199 antibody B1 and"sandwiched"by anti-CA199 antibody B7 labeled with europium chelates.A total of 90 serum samples were analysed by this new method. Results The sensitivity was 0.2 U/mL,the intra.and inter.assay CV of the Nano-TRFIA were 4.84% and 8.32% respectively,and the average recovery rate was 97.91%.The cross-reacting rates with alpha fetopro-tein and CA125 were negligible.The labeled B1 with Magnetic Microspheres was at least stable for three months at 4 ℃.Serum samples from patients and healthy blood donors were analyzed,the linear correlation of TRFIA and ECLIA measurements was positive(Y = 0.969 8X+ 4.015 3).As the gold standard of ECLIA, Nano-TRFIA had two false positive.Conclusion The newly developed Nano-TRFIA based on Magnetic Mi-crospheres technique was highly sensitive,stable and specific in the immuno-determination of serum CA199. The results showed that the methods of Nano-TRFIA based on Magnetic Microspheres could be used for the clinic.%目的 建立检测糖类抗原(CA)199的磁珠时间分辨荧光免疫分析法(Nano-TRFIA).方法 采用磁珠耦联标记抗CA199单克隆抗体(B1),Eu3+标记抗CA199单克隆抗体(Eu3+-CA199-B7),采用双抗体夹心法建立基于磁性微球法的 CA199检测方法 Nano-TRFIA,并对90份血清标本进行该方法血清学检测.结果 Nano-T RFIA检测CA199的灵敏度为0.2 U/mL,甲胎蛋白、CA125对CA199-T RFIA 均无交叉反应.磁珠耦联B14 ℃保存3个月免疫反应基本无损;同批试剂连续3个月应用分析结果稳定,样品的平均添加回收率为97.91%,批内和批间变异系数(CV)分别为4.84% 和8.32%.Nano-T RFIA 检测血清CA199结果与电化学发光法(ECLIA)检测结果显著相关(Y=0.9698 X+4.0153).以ECLIA为金标准,Nano-T RFIA 检测血清CA199时2份标本出现了假阳性.结论 Nano-T RFIA的灵敏度、特异度、准确度等均符合临床应用要求,为进一步临床验证奠定了基础.
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