Development of a multi-epitope antigen of S protein-based ELISA for antibodies detection against infectious bronchitis virus
Development of a multi-epitope antigen of S protein-based ELISA for antibodies detection against infectious bronchitis virus
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DOI：
10.1080/09168451.2015.1025692
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发表时间：
2015-08-03
影响因子：
1.6
通讯作者：
Yang, Xin
中科院分区：
文献类型：
--
作者：
Ding, Meng-die;Wang, Hong-ning;Yang, Xin
以传染性支气管炎病毒（IBV）S蛋白（SE）多表位抗原为基础，建立了检测IBV抗体的间接酶联免疫吸附试验（ELISA）。将三个S基因片段（166-247 aa，S1基因; 501-515 aa，S1基因; 8-30 aa，S2基因）串联，设计了多表位抗原SE蛋白。经Western blot分析，该融合蛋白分子量约为32 kDa，具有与IBV阳性血清结合的能力。对SE-ELISA方法的条件进行了优化。包被抗原SE的最佳浓度为3.689g/mL，采用棋盘滴定法确定一抗的稀释度为1：1000。确定截止OD 450值为0.332。SE-ELISA与IDEXX ELISA试剂盒的相对敏感性和特异性分别为92.38%和89.83%，准确性为91.46%。该方法灵敏度高，特异性强，可用于IBV抗体的检测。
An indirect enzyme-linked immunosorbent assay (ELISA) method based on a novel multi-epitope antigen of S protein (SE) was developed for antibodies detection against infectious bronchitis virus (IBV). The multi-epitope antigen SE protein was designed by arranging three S gene fragments (166-247 aa, S1 gene; 501-515 aa, S1 gene; 8-30 aa, S2 gene) in tandem. It was identified to be approximately 32kDa as a His-tagged fusion protein and can bind IBV positive serum by western blot analysis. The conditions of the SE-ELISA method were optimized. The optimal concentration of the coating antigen SE was 3.689g/mL and the dilution of the primary antibodies was identified as 1:1000 using a checkerboard titration. The cut-off OD450 value was established at 0.332. The relative sensitivity and specificity between the SE-ELISA and IDEXX ELISA kit were 92.38 and 89.83%, respectively, with an accuracy of 91.46%. This assay is sensitive and specific for detection of antibodies against IBV.