摘要
目的以纯化的重组弓形虫RH株主要表面抗原SAG1、次黄嘌呤-黄嘌呤-鸟嘌呤磷酸核糖转移酶(HXG-PRT)和腺苷激酶(AK)作为诊断抗原,建立间接ELISA法检测抗弓形虫IgG抗体。方法诱导表达本实验室保种的SAG1/pET28a/BL21、HXGPRT/pET28a/BL21和AK/pET28a/BL-21,获得rSAG1、rHXGPRT和rAK三种重组抗原。分别用不同浓度的上述抗原包被聚苯乙烯酶标板,检测疑似弓形虫感染者血清。结果混合重组抗原ELISA法的最佳rSAG1包被浓度为500ng/ml、rHXGPRT和rAK均为200ng/ml;人血清稀释度为1∶20;血清1∶200稀释仍示阳性;特异性试验的抑制率为72.36%,变异系数11.2%。结论成功表达了弓形虫三种重组诊断抗原,并应用组合抗原(rSAG1+rHXGPRT+rAK)建立了ELISA检测方法,具有高度特异性和敏感性,本试剂具有弓形虫病辅助诊断价值。
Objective Three recombinant antigens surface antigenl （SAG1）, hypoxanthine-xanthine-guanine-phos-phoribosyltransferase （HXGPRT）,and adenosine kinase（AK） of Toxoplasma gondii were evaluated for their potentiality in diagnosis of suspected patients with Toxoplasmosis, who had been screened by commercial ELISA kit. Methods The expression vectors of Toxo-SAG1/pET28a / BL21,Toxo-HXGPRT/pET28a/BL21 and Toxo-AK/pET28a/BL-21, which had been stored in our laboratory, were simultaneously induced by IPTG and identified by western blotting, rSAG1, rHXGPRT and rAK were mixed at the ratio of 1 ： 1 ： 1 and the optimal working concentrations was optimized. Combination of the three recombinant antigens were used in ELISA for the detection of sera of sixty-one suspected pregnant women, who had been screened by commercial diagnostic kit. Results Working concentrations of rSAG1, rHXGPRT and rAK antigens （ng/ml） were 500,200 and 200 ng/ml respectively. The working dilution of sera was 1 ： 20 in pregnancies. The sensitivity, specificity and accuracy in human sera, were 1 ： 200,72.36% and 11.2% respectively. Conclusion This study demonstrates that combination of the recombinant major antigens in ELISA has high sensitivity,specificity and reliability. The method is useful in the serodiagnosis of pregnancies with suspected Toxoplasma infection.
出处
《临床输血与检验》
CAS
2008年第3期239-242,共4页
Journal of Clinical Transfusion and Laboratory Medicine
关键词
弓形虫
重组抗原
酶联免疫吸附试验
免疫诊断
Toxoplasma gondii Recombinant antigen Enzyme-linked immunosorbent assay Immunodiagnosis
