夹心ELISA检测微小隐孢子虫抗原方法的建立及初步应用  

Method establishment and preliminary application of sandwich ELISA for detecting Cryptosporidium parvum antigen

作　　者：张璐[1,2] 刘珍珍 陈凯丽[1,2] 王荣军 张龙现 宁长申[1,2] 菅复春 ZHANG Lu;LIU Zhen-zhen;CHEN Kai-li;WANG Rong-jun;ZHANG Long-xian;NING Chang-shen;JIAN Fu-chun(College of Animal Science and Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002,China;International Joint Research Laboratory of Zoonosis of Henan Province,Zhengzhou 450002,China)

机构地区：[1]河南农业大学牧医工程学院,郑州450002 [2]河南省人兽共患病国际联合实验室,郑州450002

出　　处：《中国人兽共患病学报》2018年第3期230-235,247,共7页Chinese Journal of Zoonoses

基　　金：国家重点研发计划(No.2016YFD0500707);河南省科技厅基础与前沿研究项目(No.162300410166)联合资助~~

摘　　要：目的建立检测隐孢子虫的夹心ELISA方法。方法本试验以纯化的抗隐孢子虫IgG、IgY分别作为包被抗体和捕获抗体,棋盘滴定法确定夹心ELISA的最适工作条件。用基于18SrRNA的PCR方法评估饱和蔗糖溶液、饱和食盐水漂浮和PBST洗涤剂洗涤3种方法预处理样品对夹心ELISA检测效果的影响。结果建立的夹心ELISA捕获抗体、抗原、检测抗体、酶标二抗最佳工作浓度分别是1∶800、2.5μg/mL、1∶100和1∶5 000,最适捕获抗体包被、抗原抗体反应、酶标二抗反应条件分别为37℃作用1h后4℃过夜、37℃作用30min、45min;最适终止条件是2mol/L H2SO4,50μL/孔;TMB室温显色10min;特异性试验结果显示仅可以检出隐孢子虫抗原,而与常见肠道寄生的球虫卵囊、圆线虫和蛔虫虫卵无交叉反应。批间和批内变异系数均小于10%。3种方法前处理粪便用于ELISA检测与nested-PCR检测结果总符合率分别为95.83%、91.67%和83.33%,以饱和蔗糖溶液漂浮法处理检测样品的效果最佳。与爱德士的隐孢子虫检测试剂盒比较,结果显示,本方法敏感性低于爱德士试剂盒的最低检出量(6×103个/mL),整个试验过程比试剂盒多用15 min,但特异性和重复性均一致,且本试验方法更加经济实用。结论该方法简便,快速,灵敏,可用于临床粪样中隐孢子虫病原检测或隐孢子虫流行病学调查研究。We established the method of Sandwich ELISA to detect Cryptosporidium parvum antigen.Purified anti-Cryptosporidium IgG and IgY were used as a capture antibody and detection antibody respectively to develop sandwich ELISA.A checkerboard titration study was carried out to determine the optimal conditions of ELISA.The PCR based on 18SrRNA was used to evaluate the pre-treatment effect of three methods(saturated sucrose solution floating,saturated salt water floating and PBST detergent washing).The optimum concentration of coated antibody,antigen,detection antibody and enzyme-labelled antibody were 1∶800,2.5μg/mL,1∶100 and 1∶5 000 respectively.The coating condition,antigen antibody reaction,optimum reaction time of enzyme-labelled antibody were 4℃through the night after 37℃,incubated at 37℃for 30 min and 45 min respectively;the optimal termination condition was 2 mol/L H 2SO 4,50μL/well;TMB developed 10 minutes at room temperature.The developed sandwich ELISA has no cross reaction with the eggs/oocyts of Nematode,Coccidium and Ascarid;coefficient of variation of intra-assay and inter-assay were all less than 10%.The results showed that the total coincidence rate of the three pre-treatment methods with nested PCR were 95.83%,91.67%and 83.33%,respectively,and the Saturated Sucrose Floatation method was the best one among the three me thods,the sensitivity of the method was lower than the Cryptosporidium detection kit of IDEXX(6×10 3/mL),and whole test process was longer than the kit.While,its specificity and reproducibility were consistent with that of IDEXX kit,and the developed method was more economical.The method is simple,rapid,sensitive,and can be used for clinical epidemiological investigation of Cryptosporidiosis or pathogen detection.

关 键 词：隐孢子虫 双抗体夹心ELISA 检测 

分 类 号：S855.9[农业科学—临床兽医学]