新布尼亚病毒IgG抗体间接免疫荧光检测方法的建立

Establishment of indirect assay （IFA） for detection of IgG antibody against new bunyavirus

目的建立检测新布尼亚病毒IgG抗体的间接免疫荧光（IFA）方法。方法用分离到的5株新布尼亚病毒株感染非洲绿猴肾（Vero）细胞，制备抗原片，优化IFA工作条件，进行IFA法的特异度和敏感度评价，应用建立的IFA方法对2007—2011年在河南省信阳地区采集的126例发热伴血小板减少综合征患者急性期和恢复期双份血清标本进行检测，并与RT—PCR方法的检测结果进行比较。结果筛选出免疫荧光反应特异、稳定性较好的WZ69毒株作为建立新布尼亚病毒IFA检测方法的抗原种子株。IFA方法的最佳工作条件：一抗（患者血清）最适稀释度为1：40；二抗（异硫氰酸荧光素标记的羊抗人IgG抗体）最适稀释度为1：150；二抗中伊文思蓝最适稀释度为1：20000。在126例患者中，新布尼亚病毒IgG抗体阳性者96例，阴性者30例，抗体阳性率为76．19％，与RT—PCR的检测阳性率[72．22％（91／126）]差异无统计学意义（P〉0．05）。结论建立的新布尼亚病毒IgG抗体IFA检测方法敏感性高，特异性强，操作简单，可用于新布尼亚病毒致发热伴血小板减少综合征患者的实验室检测。

Objective To develop an indirect immunofluorescence assay （IFA） for detection of IgG antibodies against new bunyavirus. Methods The antigen slides were prepared with 5 new bunyavirus strains isolated using Africa green monkey kidney （Vero） cells. Specificity and sensitivity evaluation of IFA were carried out by optimizing working conditions of IFA. Using established IFA, serum samples from both acute and recovery phases were tested for 126 cases with fever thrombocytopenia and leukopenia syndrome in Xinyang, Henan province in 2007 -2011. The results were compared with detections by RT-PCR. Results The new bunyavirus stable immunofluorescenee specific WZ69 strain was selected to prepare antigen slides of IFA. The optimum conditions of IFA were： optimum dilution for primary antibody （serum） and secondary antibody （ isosulfoeyanic acid fluorescence marked goat anti-human IgG antibody） was 1 ： 40 and 1：150 respectively. The optimum dilution for Evans blue in secondary antibody was 1：20 000. Among the 126 patients, 96 paired serum specimens were tested positive to the new bunyavirus and 30 patients were tested negative to the virus. The positive rate of antibodies was 76.19%. There was no significant difference in results between IFA and RT-PCR（72.22% （91/126）） （P〉0.05）. Conclusion The IFA has high sensitivity and specificity with easy operation. It can be used in detecting the new bunyavirus infection in patients with fever, thrombocytopenia and leukopenia syndrome.