基于A1E3及B1C4单克隆抗体检测日本血吸虫循环抗原ELISA法的建立及现场初步应用

目的 目的 建立基于A1E3及B1C4单克隆抗体检测日本血吸虫循环抗原的夹心酶联免疫吸附试验 （ELISA）， 并对其应用情况进行初步评价。 方法 方法 采用十二烷基磺酸钠?聚丙烯酰胺凝胶电泳 （SDS?PAGE） 和免疫印迹试验 （Western blot?ting） 对A1E3及B1C4单克隆抗体进行特性分析， 用ELISA 法测定B1C4、 A1E3单克隆抗体效价。采用棋盘格滴定法确定双单抗夹心ELISA法检测循环抗原的最佳工作浓度。在最佳条件下， 分别检测20份急性血吸虫病病人血清、 46份慢性血吸虫病病人血清及20份正常人血清， 评价其检测敏感性和特异性。用建立的双单抗夹心ELISA法和市售检测血吸虫循环抗原的ELISA试剂盒检测湖北省江陵县IHA阳性血吸虫病病人血清72份， 以评估双单抗夹心ELISA法的检测效能。 结果 结果经SDS?PAGE和Western blotting分析， 纯化后的A1E3及B1C4在相对分子质量 （Mr ） 88 000和52 000处各有一条清晰重链，在Mr 20 000处有一条相同的轻链， 且A1E3及B1C4可与可溶性虫卵抗原 （SEA） 及急性血吸虫病病人血清发生特异性反应。B1C4单抗效价可达1∶105， A1E3单抗效价可达1∶30 000。用B1C4、 A1E3双单抗夹心ELISA法检测急、 慢性血吸虫病病人血清阳性率分别为100%和86.9%， 检测20份正常人血清特异性为100%。双单抗夹心ELISA法及市售ELISA试剂盒检测日本血吸虫循环抗原阳性率分别为45.8%及43.1%。 结论 结论 成功建立了基于A1E3及B1C4双单抗夹心ELISA法， 该法检测日本血吸虫循环抗原具有较高的敏感性及特异性。

Establishment of A1E3 and B1C4 monoclonal antibody-based ELISA for detecting circulating antigen of Schistosoma japonicum and its preliminary application

Objective To establish A1E3 and B1C4 monoclonal antibody?based ELISA for detecting circulating antigen ofSchistosoma japonicum and explore its application value in the field. Methods The characteristics of A1E3 and B1C4 monoclonalantibodies were analyzed by SDS?PAGE and Western blotting. The SEA?based ELISA was used to evaluate the titers of A1E3 andB1C4. The orthogonal test was used to determine the best concentration of coating antibody B1C4 and optimal working concentra?tion of A1E3?HRP. Under the optimal conditions，the serum samples of 20 acute schistosomiasis cases，46 chronic schistosomiasiscases，and 20 control sera were tested to evaluate its detection sensitivity and specificity. Seventy?two antibody positive serum sam?ples from Jiangling County of Hubei Province were detected and compared to a commercially available ELISA kit，to evaluate thedetection effects of this method. Results The results of SDS?PAGE demonstrated that the purified A1E3 and B1C4 contained aclear heavy chain with molecular weight of 88 000 and 52 000 respectively and had the same light chain with molecular weight of20 000；while Western blotting demonstrated that A1E3 and B1C4 could be recognized by SEA and serum samples of acute schis?tosomiasis cases. The SEA?based ELISA demonstrated the titers of B1C4 and A1E3 were 1 ∶105and 1∶30 000，respectively. Theserum samples from all the acute cases and 86.9% of the chronic cases showed a positive reaction. All of the control sera fromhealthy persons gave a negative response. The positive rates of the double monoclonal antibody ELISA and commercial ELISA fordetecting the circulating antigen were 45.8% and 43.1% respectively，and there was no significant difference between the resultsof the two methods. Conclusion A1E3 and B1C4 monoclonal antibody?based ELISA is established successfully. It exhibits a highsensitivity and specificity in detecting circulating antigen of Schistosoma japonicum.