CAJ | 학술논문

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 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。结果经SDS-PAGE和Western blotting分析，纯化后的A1E3及B1C4在相对分子质量（Mr）88000和52000处各有一条清晰重链，在Mr 20000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∶30000。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。
목적：건립기우A1E3급B1C4단극륭항체검측일본혈흡충순배항원적협심매련면역흡부시험（ELISA），병대기응용정황진행초보평개。방법채용십이완기광산납-취병희선알응효전영（SDS-PAGE）화면역인적시험（Western blot-ting）대A1E3급B1C4단극륭항체진행특성분석，용ELISA법측정B1C4、A1E3단극륭항체효개。채용기반격적정법학정쌍단항협심ELISA법검측순배항원적최가공작농도。재최가조건하，분별검측20빈급성혈흡충병병인혈청、46빈만성혈흡충병병인혈청급20빈정상인혈청，평개기검측민감성화특이성。용건립적쌍단항협심ELISA법화시수검측혈흡충순배항원적ELISA시제합검측호북성강릉현IHA양성혈흡충병병인혈청72빈，이평고쌍단항협심ELISA법적검측효능。결과경SDS-PAGE화Western blotting분석，순화후적A1E3급B1C4재상대분자질량（Mr）88000화52000처각유일조청석중련，재Mr 20000처유일조상동적경련，차A1E3급B1C4가여가용성충란항원（SEA）급급성혈흡충병병인혈청발생특이성반응。B1C4단항효개가체1∶105，A1E3단항효개가체1∶30000。용B1C4、A1E3쌍단항협심ELISA법검측급、만성혈흡충병병인혈청양성솔분별위100%화86.9%，검측20빈정상인혈청특이성위100%。쌍단항협심ELISA법급시수ELISA시제합검측일본혈흡충순배항원양성솔분별위45.8%급43.1%。결론성공건립료기우A1E3급B1C4쌍단항협심ELISA법，해법검측일본혈흡충순배항원구유교고적민감성급특이성。
Objective To establish A1E3 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 A1E3 and B1C4 monoclonal antibodies were analyzed by SDS-PAGE and Western blotting. The SEA-based ELISA was used to evaluate the titers of A1E3 and B1C4. 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 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 effects of this method. Results The results of SDS-PAGE demonstrated that the purified A1E3 and B1C4 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 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∶105 and 1∶30 000,respectively. The serum samples from all the acute cases and 86.9% of the chronic cases showed a positive reaction. All of the control sera from healthy 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 A1E3 and B1C4 monoclonal antibody-based ELISA is established successfully. It exhibits a high sensitivity and specificity in detecting circulating antigen of Schistosoma japonicum.