预警哨鼠抗Sj23HD IgG抗体Western blot检测方法的改进及应用

Improvement and use of Western blotting to detect IgG antibodies against Sj23HD for early diagnosis of schistosomiasis in sentinel mice

目的改进检测预警哨鼠血清抗日本血吸虫23ku膜蛋白大亲水肽段（Sj23HD）IgG抗体的Western blot方法，并观察其现场应用价值。方法预先制备转印有重组si23HD蛋白的NC膜条，采用化学发光Western blot检测哨鼠血清抗sj23HD IgG，观察方法的稳定性和敏感性，并对2012年江苏省潜在血吸虫感染高风险水域监测预警哨鼠回收后第14、21、35d血样进行检测，同时以ELISA检测方法作为对照，对免疫检测结果与解剖查虫结果进行统计学分析。结果改进后的Western blot操作简便，比常规方法节省142周时间。预制NC膜条至少可低温保存6个月，其敏感性与新鲜制备的NC膜条的敏感性相似。使用改进后的Western blot方法检测预警哨鼠14、21和35d血清标本，阳性率分别为0．90％，3．50％和5．06％，ELISA阳性率分别为0．45％、3．00％和5．06％，差异无统计学意义（P〉0．05）。两种免疫学方法的敏感性均高于解剖查虫法。结论改进后的Western blot操作简便，敏感性高，可用于预警哨鼠血吸虫感染检测。

Objective To improve Western blotting to detect specific IgG antibodies against the large hydrophilic domain （HD） of the 23-ku membrane protein of Schistosoma japonicum （Sj23HD） to monitor early schistosomiasis in sentinel mice and to determine the value of its actual use. Methods NC membranes with the Sj23HD protein attached were pre- pared ahead of time and chemiluminescence was used to detect specific IgG antibodies against Sj23HD antigen in sera from sentinel mice. The consistency and sensitivity of this Western blotting were determined. Sentinel mice were exposed to a water area with a high prevalence of schistosome cercariae in Jiangsu Province in 2012, and the sera of these mice were tested with Western blotting on days 14, 21, and 35 after exposure. These serum samples were also tested with ELISA to serve as a control. Results of both immunological tests were compared to the results of the worm count upon dissection. Results Improved Western blotting was simpler than conventional methods, saving one or even two weeks of time. The NC membrane strips prepared ahead of time were stored at a low temperature for at least six months with no change in sensitivity. Sentinel mice were collected. Simplified Western blotting indicated that their sera tested positive for IgG antibodies against Sj23HD at a rate of 0.9% on day 14, at a rate of 3.5% on day 21, and at a rate of 5.06% on day 35 while ELISA indicated that their sera tested positive for those antibodies at a rate of 0.4, 3.0, and 5.06 %. Differences in the rate of detection were not statistically significant （P〈0.05）. Both immnnological methods had a higher sensitivity than that of the worm count upon dissection. Conclusion Improved Western blotting has a high sensitivity and is easy to perform, allowing early detection of schistosomiasis in sentinel mice.