新型布尼亚病毒抗原ELISA检测方法建立及初步评价

Establishment and Evaluation of ELISA for Detecting Novel Bunyavirus Antigen

目的 建立发热伴血小板减少综合征布尼亚病毒抗原检测方法,并进行初步临床评价.方法 克隆表达新型布尼亚病毒核蛋白（NP）,分别免疫家兔和小鼠,制备多抗和单抗,以单抗包被酶联反应板,多抗偶联辣根过氧化物酶（HRP）,建立双抗体夹心免疫酶联方法,将获得的NP蛋白进行系列稀释,制作标准曲线,评价其检测灵敏度.分别检测48份临床发热伴血小板减少综合征和48份健康献血员血清.结果 建立的发热伴血小板减少综合征布尼亚病毒抗原检测方法检测灵敏度为1 pg/ml,48份临床疑似病人检出阳性7例（14.58%）,48份健康血清样品1例阳性,特异度98%.结论 建立发热伴血小板减少综合征新型布尼亚病毒抗原检测方法可用于发热伴血小板减少综合征新型布尼亚病毒早期检测.

Objective To do establishment and evaluation of an enzyme-linked immunosorbent assay （ELISA） kit for detec-ting novel bunyavirus antigen of fever with thrombocytopenia associated syndrome. Methods The vector pET30a was used to clone and express the antigens of novel bunyavirus （NP）. According to the kit,a rabbit and a mouse were immunized by using NP antigen. An enzyme-linked reaction plate was coated by using monoclonal antibody, and rabbit antibody was cou- pled with peroxidase （HRP）. The establishment of ELISA kit for detecting novel bunyavirus antigen. The serial dilution of antigen was used to make standard curve for the evaluating detectablity. The healthy blood donation 48 and fever with thrombocytopenia associated syndrome 48 were detected. Results The novel bunyavirus （NP） protein and monoclonal anti-body,rabbit antibody was obtained. An ELISA kit for detecting novel bunyavirus antigen was established and the results showed that the detecting sensitivity was lpg/ml. The specificity was 98% in 48 healthy blood donation and positive 7 in 48 fever with thrombocytopenia associated syndrome. Conclusion An ELISA kit established is used for detecting novel bunya-virus antigen,which provide useful tools for diagnosis and analyzing of fever with thrombocytopenia associated syndrome.