摘要
目的比较弓形虫rSAG1、rHXGPRT和rAK三种重组蛋白作为混合抗原在弓形虫病血清免疫诊断中检测结果与进口试剂盒检测结果的差异,评价三种重组蛋白混合抗原检测在弓形虫病免疫诊断中的应用价值。方法收集经美国产Zeus Toxo-ELISA试剂盒筛选的阳性血清136例,阴性血清30例。复苏、诱导、表达含有弓形虫pET-28a/SAG1,pET-28a/HXGPRT和pET-28a/AK表达载体的3个菌株,纯化3种重组蛋白。以3种混合蛋白作为诊断抗原,采用间接ELISA法,以正交叉方法优化检测条件,检测66例弓形虫IgM阳性、70例IgG例阳性血清和30阴性血清,比较混合抗原检测和Zeus Toxo-ELISA试剂盒检测结果的差异;以Zeus Toxo-ELISA弓形虫试剂盒检测结果为标准,评价重组蛋白混合抗原检测的敏感性、特异性、总符合率以及约登指数。结果 IgM:混合抗原与Zeus Toxo-ELISA试剂检测结果经χ2检验,χ2值为3.12,P>0.05,差异无显著性。混合重组抗原的敏感性为89.4%,特异性为96.7%,总符合率为91.7%,约登指数为0.861。IgG:混合抗原与Zeus Toxo-ELISA试剂检测结果经χ2检验,χ2值为0.25,差异无显著意义(P>0.05)。采用混合重组抗原的敏感性为97.1%,特异性为93.3%,总符合率为96.0%,约登指数为0.904。结论本研究3种重组抗原(rSAG1+rHXGPRT+rAK)混合的间接ELISA法检测弓形虫IgM和IgG,与进口ELISA试剂检测的总符合率高,具有较高的敏感性、特异性;更经济。
ABSTRACT：The efficacy of immunodiagnosis of Toxoplasma infection by using recombinant antigens of rSAG1, rHXG- PRT and rAK and the imported commercial reagent kit （Zeus Toxo-ELISA） was evaluated in this study. One hundred and thir- ty-six cases of positive sera and 30 cases of negative sera that had been detected with imported kit were subjected to the detec- tion of （rSAG1 ＋ rHXGPRT＋ rAK）-ELISA. The three expression vectors of Toxo-SAG1/pET-28a/BL21, Toxo-HXGPRT/ pET-28a/BL21, and Toxo-AK/pET-28a/BL21 previously generated in the laboratory were induced with isopro pyl-beta-D-thio- gactoside （IPTG）, followed by purification through affinity chromatography with Niz＋. After optimization, rSAG1, rHXG- PRT and rAK were used as the coating antigens for detection of 66 cases of IgM positive sera, 70 cases of IgG positive sera and 30 negative sera of Toxoplasrna, which had been screened by the commercial diagnostic kit, were tested with indirect ELISA. The results of the two methods were analyzed. It showed that in 66 cases of IgM positive sera detected with the commercial kit, the positive rate was 89.40%, the negative rate was 96.7 %, and the Youden index was 0. 861 （X2= 3.12, P〉0.05） when test- ed with （rSAG1 ＋ rHXGPRT＋ rAK）-ELISA method. No statistic difference was found between the two methods. Correspond-ingly, in 70 cases of IgG positive sera screened with the commercial kit, the （rSAG1 q- rHXGPRT ＋ rAK）-ELISA method gave rise to a positive rate of 97. 1% and a negative rate of 93.3%. No significant difference was noted between the two methods （X2=0.25, P〉0. 05）. In conclusion, the （rSAG1 -b rHXGPRT q- rAK）-ELISA has a promising coin-cidence with the imported Toxo-ELISA kit with high sensitivity and specificity.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2014年第4期358-363,共6页
Chinese Journal of Zoonoses
基金
国家重大基础研究计划资助(973计划
No.2010CB530001)
安徽省教育厅自然科学基金资助(No.KJ2008B311)~~
