蓝舌病病毒双抗体夹心ELISA检测方法的建立

Development of double antibody sandwich ELISA for detection of bluetongue virus

用将纯化的蓝舌病病毒(BTV)免疫BALB/c小鼠,分离免疫鼠脾细胞,与SP2/0细胞融合,经间接ELISA筛选,得到了3株(1F5、4E5和6A1)可以稳定分泌抗BTV VP7特异性单抗的杂交瘤细胞株。用纯化的BTV免疫家兔,按常规方法制备BTV多抗。选用抗VP7单抗(1F5)包被ELISA板,用兔抗BTV多抗作为捕获抗体,建立了BTV双抗体夹心ELISA检测方法。用该ELISA方法分别检测BTV、鹿流行性出血病病毒、水疱性口炎病毒、赤羽病病毒、小反刍兽疫病毒阳性样品。结果表明,该ELISA方法具有良好的特异性。用该ELISA和RT-PCR同时检测259份临床样品,ELISA的特异性和敏感性分别为99.1%和85.7%,两种方法的符合率为97.7%。该方法的建立为BTV的检测及蓝舌病的流行病学调查提供了有效工具。

Six-week-old SPF BALB/c mice were immunized with purified bluetongue virus（BTV） emulsified with Freund＇s adjuvant. The spleen cells isolated from vaccinated mice were fused with SP2/0 cells. Three hybridoma of 1F5,4E5 and 6A1, screened using ELISA, BTV VP7-ELISA and BHK-ELISA could secrete stably specific antibodies to VP7 protein of BTV. A double antibody sandwich ELISA（DAS- ELISA） for detection of BTV was established based on monoclonal antibody（1FS） and rabbit-anti-BTV polyclonal antibody. Positive samples with BTV or epizootic haemorrhagic disease virus of deer, vesicular stomatitis virus, Akabane disease virus and Peste des petits ruminants virus were examined by the estab- lished ELISA, respectively. The result showed that DAS-ELISA had good specificity. Then DAS-ELISA was used to detect 259 clinical samples,and RT-PCR was used as a reference method. The results showed that the specificity and sensitivity of DAS-ELISA were 99.1% and 85.7%, respectively, and the agreement ratio of DAS-ELISA to RT-PCR was 97. 7%. These results indicated that DAS-ELISA was suitable for rapid detection and epidemiological investigation of BTV infection in ruminants.