双抗体夹心ELISA检测旋毛虫循环抗原实验条件的研究  

Experimental conditions for double antibody sandwich ELISA to detect circulating Trichinella spiralis antigens

作　　者：刘莉娜[1] 崔晶[1] 甘冠华[1] 潘亮[1] 陈琳[1] 乔呈瑞[1] 王中全[1] 

机构地区：[1]郑州大学医学院寄生虫学教研室,河南郑州450052

出　　处：《中国病原生物学杂志》2013年第5期423-425,共3页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.30972492;81141084;30972579);郑州大学大学生创新计划项目(No.2012cxsy137)

摘　　要：目的建立检测旋毛虫循环抗原(circulating antigens,CAg)的双抗体夹心ELISA方法。方法以抗旋毛虫肌幼虫排泄-分泌(excretory-secretory,ES)抗原的鸡卵黄抗体IgY作为包被抗体,以抗ES抗原的小鼠单抗IgM作为检测抗体,建立监测旋毛虫CAg的双抗体夹心ELISA方法;应用棋盘滴定法,选择包被抗体、待测血清、检测抗体及酶结合物的最佳稀释度,并测定方法的敏感性。结果双抗体夹心ELISA检测旋毛虫CAg的最佳条件为:IgY包被浓度为7.5μg/ml,血清稀释度为1︰60,单抗稀释度为1︰2000,辣根过氧化物酶标记羊抗小鼠IgM抗体稀释度为1︰2000。该方法检测旋毛虫CAg的敏感性为1ng/ml。结论建立的旋毛虫CAg双抗体夹心ELISA法具有较高的敏感性,有望用于旋毛虫病的早期诊断。Objectives To establish double antibody sandwich ELISA to detect circulating Trichinella spiralis antigens （CAg）. Methods Chick egg yolk IgY antibodies against the excretory-secretory （ES） antigens of T. spiralis muscle larvae were used as the capturing antibody and mouse IgM monoclonal antibody （mAb） was used as detecting antibody. The optimal dilutions of the capture antibodies, serum samples, detecting antibodies, and IgM-HRP were determined by using chessboard titration. The sensitivity of sandwich ELISA at detecting T. spiralis CAg was also assayed. Results The optimal conditions for sandwich ELISA to detect T. spiralis CAg were as follows： concentration of IgY for coating of 7.5 μg/ml, a diluted serum concentration of 1：60, a diluted concentration of IgM mAb of 1：2 000, and a diluted concentration of HRP-conjugated sheep anti-mouse IgM of 1：2 000. The sensitivity of the sandwich ELISA for detecting T. spiralis CAg was 1 ng/ml. Conclusion A novel technique for double antibody sandwich ELISA as was established in this study is a promising way to diagnose trichinellosis early.

关 键 词：旋毛虫 循环抗原 IGY 单克隆抗体 夹心ELISA 

分 类 号：R383.15[医药卫生—医学寄生虫学]