Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, China.
Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, Chengdu 610064, China.
Int J Mol Sci. 2022 Jul 8;23(14):7589. doi: 10.3390/ijms23147589.
Avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is the causative agent of infectious bronchitis (IB) that has brought great threat and economic losses to the global poultry industry. Rapid and accurate diagnostic methods are very necessary for effective disease monitoring. At the present study, we screened a novel nanobody against IBV-N protein for development of a rapid, simple, sensitive, and specific competitive ELISA for IBV antibody detection in order to enable the assessment of inoculation effect and early warning of disease infection. Using the phage display technology and bio-panning, we obtained 7 specific nanobodies fused with horseradish peroxidase (HRP) which were expressed in culture supernatant of HEK293T cells. Out of which, the nanobody of IBV-N-Nb66-vHRP has highly binding with IBV-N protein and was easily blocked by the IBV positive serums, which was finally employed as an immunoprobe for development of the competitive ELISA (cELISA). In the newly developed cELISA, we reduce the use of enzyme-conjugated secondary antibody, and the time of whole operation process is approximately 1 h. Moreover, the IBV positive serums diluted at 1:1000 can still be detected by the developed cELISA, and it has no cross reactivity with others chicken disease serums including Newcastle disease virus, Fowl adenovirus, Avian Influenza Virus, Infectious bursal disease virus and Hepatitis E virus. The cut-off value of the established cELISA was 36%, and the coefficient of variation of intra- and inter-assay were 0.55-1.65% and 2.58-6.03%, respectively. Compared with the commercial ELISA (IDEXX kit), the agreement rate of two methods was defined as 98% and the kappa value was 0.96, indicating the developed cELISA has high consistency with the commercial ELISA. Taken together, the novel cELISA for IBV antibody detection is a simple, rapid, sensitive, and specific immunoassay, which has the potential to rapidly test IBV antibody contributing to the surveillance and control of the disease.
禽冠状病毒-传染性支气管炎病毒（AvCoV-IBV）是传染性支气管炎（IB）的病原体，给全球家禽业带来了巨大的威胁和经济损失。快速准确的诊断方法对于有效的疾病监测非常必要。在本研究中，我们筛选了一种针对 IBV-N 蛋白的新型纳米抗体，用于开发一种快速、简单、灵敏、特异的竞争 ELISA 方法，用于检测 IBV 抗体，以评估接种效果和疾病感染的预警。我们使用噬菌体展示技术和生物淘选，从 HEK293T 细胞的培养上清液中获得了 7 种与辣根过氧化物酶（HRP）融合的特异性纳米抗体。其中，IBV-N-Nb66-vHRP 纳米抗体与 IBV-N 蛋白具有高度结合性，并且容易被 IBV 阳性血清阻断，最终被用作竞争 ELISA（cELISA）的免疫探针。在新开发的 cELISA 中，我们减少了酶结合的二抗的使用，整个操作过程的时间约为 1 小时。此外，用建立的 cELISA 可以检测稀释度为 1:1000 的 IBV 阳性血清，并且与其他鸡病血清如新城疫病毒、禽腺病毒、禽流感病毒、传染性法氏囊病病毒和戊型肝炎病毒无交叉反应。该 cELISA 的临界值为 36%，批内和批间变异系数分别为 0.55-1.65%和 2.58-6.03%。与商业 ELISA（IDEXX 试剂盒）相比，两种方法的符合率定义为 98%，kappa 值为 0.96，表明建立的 cELISA 与商业 ELISA 具有高度一致性。综上所述，该新型 cELISA 用于 IBV 抗体检测是一种简单、快速、灵敏、特异的免疫分析方法，具有快速检测 IBV 抗体的潜力，有助于疾病的监测和控制。
