免疫-PCR检测肾综合征出血热患者血清中抗核衣壳蛋白抗体方法的建立

Establishment of immune-PCR technique for the detection of anti-mucleocapsid protein antibodies in sera of patients with hemorrhagic fever associated with renal syndrome

目的 建立高灵敏性和高特异性的免疫 -PCR方法检测肾综合征出血热 (HFRS)患者血清中抗核衣壳蛋白抗体。方法 用汉坦病毒 ( 76 - 118株 )重组核衣壳蛋白为抗原包被聚苯乙烯微滴度板 ,与经ELISA法和临床均已确诊为HFRS患者的血清进行抗原抗体特异性反应 ,以链霉亲和素为桥梁将生物素标记的抗体与生物素标记的DNA指示分子相连 ,PCR扩增DNA指示分子 ,通过检测指示分子DNA来反映肾综合征出血热患者血清中抗核衣壳蛋白抗体的水平 ,并与传统的ELISA法进行比较。结果 免疫 -PCR检测肾综合征出血热患者血清中抗核衣壳蛋白抗体的敏感性是ELISA法的 2 5 0 0 0倍 ;30例HFRS阳性患者血清免疫 -PCR检测均为阳性 ,2 0例甲型肝炎阳性血清和 2 0例正常人血清免疫 -PCR检测均为阴性。结论 免疫 -PCR方法敏感性高 ,特异性强 。

To establish a highly sensitive and specific method to detect the anti nucleocapsid protein antibodies in sera of patients with hemorrhagic fever as sociated with renal syndrome(HFRS), the recombinant nucleocapsid protein (NP) of Hantaan virus(strain 76 118) was coated on the polycarbonate microtiter plates,and reacted specifically with sera from the HFRS patients diagnosed clinically and proved by ELISA assay.Streptavdin was used to bridge the biotinylated antibodies and biotinylated DNA, which was amplified by PCR as marker molecule.The relevant anti NP antibodies in sera were indicated by detection of the marker molecule.This modified immuno PCR method was compared with the conventional ELISA assay at the same time.It was found that the sensitivity of immuno PCR method was 25000 times higher than that of the conventional ELISA,in which all the positive sera of 30 cases with HFRS were proved to be positive by immuno PCR method;while all the positive sera from 20 cases of HAV infection and 20 normal human sera were proved to be negative by immuno PCR method.These results indicates that the immuno PCR method is quite sensitive and specific,and can be used as a powerful and very sensitive tool for the clinical diagnosis of HFRS.