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

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

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 blotting)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Mr20 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。

Objective To establish AlE3 and B1C4 monoclonal antibody-based ELISA for detecting circulating antigen of Schistosoma japonicum and explore its application value in the field. Methods The characteristics of A 1 E3 and B 1 C4 monoclonal antibodies were analyzed by SDS-PAGE and Western blotting. The SEA-based ELISA was used to evaluate the titers of A I E3 and B 1 C4. The orthogonal test was used to deterxnine the best concentration of coating antibody B I C4 and optimal working eoneentra lion of A 1E3-HRP. Under the optimal conditions, the serum samples of 20 acute schistosomiasis cases, 46 chronic schistosomiasis cases, 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 the detection effectts of this method. Results The results of SDS-PAGE demonstrated that the purified AlE3 and BIC4 contained a clear heavy chain with molecular weight of 88 000 and 52 000 respectively and had the same light chain with molecular weight of 20 000; while Western blotting demonstrated that AlE3 and B1C4 could be recognized by SEA and serum samples of acute schis tosomiasis eases. The SEA-based EL1SA demonstrated the tilers of BIC4 and AlE3 were l ： l0s and 1 ： 30 000, respectively. The serum samples from all the acute cases and 86.9% of the chronic eases 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 for detecting the circulating antigen were 45.8% and 43.1% respectively, and there was no significant difference between the results of the two methods. Conclusion AlE3 and B1C4 monoclonal antibody-based ELISA is established successfully. It exhibits a high sensitivity and specificity in detecting circulating antigen of Schistosoma japonicum.