Yang Xue, Ye Yan, Wang Tingting, Li Mei, Yu Lei, Xia Min, Qian Jun, Hu Zhigang
Affiliated Wuxi People's Hospital of Nanjing Medical University, Wuxi Children's Hospital, Wuxi, Jiangsu, China.
J Clin Lab Anal. 2019 Feb;33(2):e22659. doi: 10.1002/jcla.22659. Epub 2018 Aug 26.
To develop a new immunoassay based on the time-resolved fluorescence immunoassay (TRFIA) system for the simultaneous measurement of IgM and IgG antibodies to HCV.
Coated recombinant HCV antigens and Eu -labeled IgM and Sm -labeled IgG antibodies were prepared. HCV-IgM/IgG TRFIA was established and optimized, followed by a methodological assessment. Data were expressed as +SD and analyzed using the SPSS 13.0 software. The percentile method was used to calculate cutoff values.
The detection sensitivities of HCV-IgM and HCV-IgG were 0.06 S/CO and 0.15 S/CO, respectively. There was a good linear response from 1:40 to 1:40 960 for HCV-IgM and 1:20 to 1:40 960 for HCV-IgG, when samples strongly positive for HCV-IgM and HCV-IgG were serially diluted from 1:10 to 1:81 920. The average intra-assay coefficients of variation (CV) for HCV-IgM and -IgG were 3.45% and 3.71% and the inter-assay coefficients of variation (CV) were 6.49% and 6.79%, respectively. When HCV-negative/positive sera were tested by ELISA and using established kits, the negative, positive, and total conformity rates for HCV-IgM were 93.3% (28/30), 100% (25/25), and 96.4% (53/55), and those for HCV-IgG were 93.3% (28/30), 100% (35/35), and 96.9% (63/65), respectively. Additionally, the established kit exhibited good stability, with declines in fluorescence values to 11.1% and 9.5%, respectively, after storage at 37°C for 7 days.
We established a dual-label HCV-IgM/IgG TRFIA assay with a wide detection range, high specificity, high sensitivity, good stability, and good clinical value for the simultaneous measurement of HCV-IgM and HCV-IgG titers in a single test.
开发一种基于时间分辨荧光免疫分析（TRFIA）系统的新型免疫分析法，用于同时检测抗丙型肝炎病毒（HCV）IgM和IgG抗体。
制备包被重组HCV抗原以及铕标记的IgM和钐标记的IgG抗体。建立并优化HCV-IgM/IgG TRFIA，随后进行方法学评估。数据以均值±标准差表示，并使用SPSS 13.0软件进行分析。采用百分位数法计算临界值。
HCV-IgM和HCV-IgG的检测灵敏度分别为0.06 S/CO和0.15 S/CO。当HCV-IgM和HCV-IgG强阳性样本从1:10连续稀释至1:81 920时，HCV-IgM在1:40至1:40 960、HCV-IgG在1:20至1:40 960范围内呈现良好的线性反应。HCV-IgM和IgG的批内变异系数（CV）平均值分别为3.45%和3.71%，批间变异系数（CV）分别为6.49%和6.79%。用ELISA法及已建立的试剂盒检测HCV阴性/阳性血清时，HCV-IgM的阴性、阳性及总符合率分别为93.3%（28/30）、100%（25/25）和96.4%（53/55），HCV-IgG的分别为93.3%（28/30）、100%（35/35）和96.9%（63/65）。此外，所建立的试剂盒稳定性良好，在37°C保存7天后荧光值分别下降至11.1%和9.5%。
我们建立了一种双标记HCV-IgM/IgG TRFIA检测方法，该方法检测范围广、特异性高、灵敏度高、稳定性好，在单次检测中同时测定HCV-IgM和HCV-IgG滴度具有良好的临床价值。
