摘要目的 建立能同时检测抗HCV-免疫球蛋白(Ig)M和抗HCV-IgG抗体的时间分辨荧光免疫分析(TRFIA)法.方法 采用HCV重组抗原包被,制备Eu3+标记抗人IgM和Sm3+标记抗人IgG抗体;采用间接法建立HCV-IgM和HCV-IgG TRFIA并行优化和方法学考核.用百分位数法单侧95％计算阳性判定(CO)值.结果 以1个样本检测值/阳性判定值(S/CO)为单位,HCV-IgM和HCV-IgG的检测灵敏度分别为0.06和0.15 S/CO.分别将HCV-IgM和HCV-IgG强阳性的标本从1:12.5倍比稀释到1:51200,HCV-IgM在1:12.5～1:12800呈较好线性反应,HCV-IgG在1:25～1:6400呈较好线性反应.HCV-IgM和HCV-IgG的平均批内CV分别为3.37％和3.66％,平均批间CV分别为6.52％和6.75％.用酶联免疫吸附测定法作为参照,检测HCV阳性标本和阴性标本各20例,两者HCV-IgM阳性符合率为100.0％(20/20),阴性符合率为90.0％(18/20),总符合率为95.0％(38/40);HCV-IgG相应数据分别为100.0％(20/20)、95.0％(19/20)和97.5％(39/40).试剂盒于37℃ 放置7 d检测,荧光计数值分别下降11.1％和9.5％,稳定性能良好.结论 建立的HCV-IgM和HCV-IgG TRFIA双标记分析法1次测量可同时得到HCV-IgM和HCV-IgG结果,是一种宽量程、高特异性、高灵敏度、稳定性好的方法.

abstractsObjective To establish a time-resolved fluoroimmunoassay (TRFIA) system for simul-taneous measurement of immunoglobulin (Ig)M and IgG antibodies to HCV. Methods Recombinant HCV antigen was fixed on microtiter plates to detect serum HCV antibodies. Eu3+-labeled anti-human IgM and Sm3+-labeled anti-human IgG were prepared. HCV-IgM and HCV-IgG TRFIA were established using indirect assay and further optimized and evaluated. The one-sided 95th-percentile was used to calculate the cut-off (CO) values. Results Defining 1 sample/ CO (S/ CO) as measuring unit, the detection limit was 0.06 S/ CO for HCV-IgM and 0.15 S/ CO for HCV-IgG. When diluted a strong-positive specimen from 1 :12.5 to 1 :51200, there was a good liner range within 1 :12.5 to 1 :12800 for HCV-IgM and 1 :25 to 1 :6400 for HCV-IgG. The average intra-assay CV of HCV-IgM and HCV-IgG were 3.37％ and 3.66％, respectively, and the aver-age inter-assay CV were 6.52％ and 6.75％, respectively. Compared with enzyme-linked immunosorbent as-say (ELISA) kits, the positive conformity rate, the negative conformity rate and total conformity rate were 100.0％(20/ 20), 90.0％(18/ 20), 95.0％(38/ 40) for HCV-IgM TRFIA, and were 100.0％ (20/ 20), 95. 0％(19/ 20), 97.5％(39/ 40) for HCV-IgG TRFIA, respectively. Additionally, the established HCV-IgM and HCV-IgG assay kits presented good stability with a decline in the value of fluorescence of 11.1％and 9.5％ respectively after being stored at 37 ℃ for 7 d. Conclusions The established HCV-IgM and HCV-IgG TRFIA could simultaneously measure HCV-IgM and HCV-IgG antibodies at one step. The method has wider detectable range and may be a more sensitive, stable, and reliable method for diagnosing HCV in-fection.