双抗夹心ELISA法检测弓形虫核苷三磷酸脱氢酶-Ⅱ型蛋白的研究  

Application of Double Antibody Sandwich ELISA for Detection of Nucleoside Triphosphate Hydrolase-Ⅱ Protein of Toxoplasma gondii

作　　者：胡昕[1] 诸葛青云[2] 李亚飞[1] 潘长旺[2] 谭峰[2] 

机构地区：[1]温州医学院生命科学学院、检验医学院,温州325035 [2]温州医学院基础医学院,温州325035

出　　处：《中国寄生虫学与寄生虫病杂志》2010年第5期343-347,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：浙江省科技厅面上社会发展项目(No2009C33035)~~

摘　　要：目的建立双抗夹心ELISA法检测弓形虫核苷三磷酸脱氢酶-Ⅱ型(NTPase-Ⅱ)蛋白。方法用重组弓形虫NTPase-Ⅱ(rTgNTPase-Ⅱ)蛋白免疫BALB/c小鼠,筛选高滴度、高特异性的单克隆抗体建立双抗夹心ELISA法。通过检测弓形虫速殖子全虫蛋白与rTgNTPase-Ⅱ的浓度评价该方法的敏感性。通过对疟疾(7例)、日本血吸虫病(12例)、并殖吸虫病(14例)和脑囊尾蚴病(10例)患者血清进行交叉反应试验,评价该方法的特异性。结果获得2株稳定分泌抗rTgNTPase-Ⅱ蛋白单克隆抗体的杂交瘤细胞株,命名为MNT1和MNT2。蛋白质印迹分析(Westernblotting)显示,2株单克隆抗体均能特异性识别弓形虫rTgNTPase-Ⅱ蛋白和弓形虫速殖子全虫蛋白。以MNT1为包被抗体,MNT2为酶标抗体,建立的双抗夹心ELISA可检测出全虫蛋白的最低浓度为6μg/ml,检测出rTgNTPase-Ⅱ的最低浓度为1.5μg/ml。该方法的特异性为100%。结论以抗rTgNTPase-Ⅱ蛋白单克隆抗体MNT1与MNT2为基础建立的双抗夹心ELISA法具有较高的特异性。Objective To establish a double antibody sandwich ELISA method for detection of nucleoside triphos- phate hydrolase-Ⅱ （NTPase-Ⅱ） protein of Toxoplasma gondii. Methods BALB/c mice were immunized with recombinant NTPase-Ⅱ （rTgNTPase-Ⅱ） protein of T. gondii. The hybridomas that secreted high titer of monoclonal antibodies （mAbs） with high specificity were screened and used to establish the double antibody sandwich ELISA for the detection of rTgNTPase-Ⅱ. In order to evaluate the sensitivity of the method, the concentration of whole-tachyzoite lysate and rTgNTPase- Ⅱ was detected, respectively. Serum samples from patients with malaria （7 cases）, schistosomiasis （12 cases）, paragoni- miasis （14 cases） and cysticercosis （10 cases） were examined by the same method. Results Two hybridoma cell lines, MNT1 and MNT2, were developed for secreting mAbs against rTgNTPase-Ⅱ. Western blotting analysis showed that the two mAbs specifically recognized rTgNTPase-Ⅱ and whole-tachyzoite lysate. The MNT1 was used as coating antibody, and HRP-labeled MNT2 as secondary antibody. The double antibody sandwich ELISA detecting rTgNTPase-Ⅱ was developed with a minimum concentration of 6 μg/ml for whole-tachyzoite lysate and 1.5 μg/ml for rTgNTPase-Ⅱ. An overall specificity of 100% was determined. Conclusion The double antibody sandwich ELISA based on MNT1 as coating antibody and MNT2 as secondary antibody has a high specificity.

关 键 词：刚地弓形虫 单克隆抗体 核苷三磷酸脱氢酶-Ⅱ型蛋白 双抗夹心ELISA 

分 类 号：R531.8[医药卫生—内科学]