禽传染性支气管炎病毒(IBV)仍然在养禽业造成严重的经济损失。目前,IBV有多种流行的基因型和血清型。开发一种新的快速诊断方法势在必行,敏感,具体,简单,和广谱。单克隆杂交瘤细胞,在骨髓瘤SP2/0细胞与从免疫的Balb/c小鼠分离的脾细胞融合后,获得针对IBVN蛋白的N2D5。N2D5单克隆抗体(mAb)和先前制备的针对IBVN蛋白的小鼠多克隆抗体用于靶向IBV,作为胶体金-mAb缀合物和捕获的抗体,分别,以开发免疫层析试纸。胶体金-mAbN2D5偶联反应体系的最佳pH值和最小抗体浓度分别为pH6.5和30μg/mL,分别。测试了常见的禽类病原体以评估条带的特异性,并且没有观察到交叉反应。试纸检测IBV的灵敏度为10-1.4522EID50/mL。该试纸显示出检测IBV抗原的广谱交叉反应能力,包括中国的多种IBV基因型和目前在中国南方流行的所有7种IBV血清型。此外,在没有任何设备的情况下,可以在2分钟内观察到结果。使用开发的试纸和qPCR方法对人工感染了三种IBV菌株的鸡的喉咙和泄殖腔拭子样品进行了测试;试纸在通过qPCR基因检测IBV方面表现出很高的一致性。总之,建立的免疫层析试纸是快速的,敏感,具体,简单,实用,和广谱;此外,它有潜力作为IBV的现场快速检测方法,可以促进疾病的监测和控制,特别是在资源有限的地区。
Avian infectious bronchitis virus (IBV) still causes serious economic losses in the poultry industry. Currently, there are multiple prevalent genotypes and serotypes of IBVs. It is imperative to develop a new diagnosis method that is fast, sensitive, specific, simple, and broad-spectrum. A monoclonal hybridoma cell, N2D5, against the IBV N protein was obtained after fusion of myeloma SP2/0 cells with spleen cells isolated from the immunized Balb/c mice. The N2D5 monoclonal antibody (mAb) and the previously prepared mouse polyclonal antibody against the IBV N protein were used to target IBV as a colloidal gold-mAb conjugate and a captured antibody, respectively, in order to develop an immunochromatographic strip. The optimal pH and minimum antibody concentration in the reaction system for colloidal gold-mAb N2D5 conjugation were pH 6.5 and 30 μg/mL, respectively. Common avian pathogens were tested to evaluate the specificity of the strip and no cross-reaction was observed. The sensitivity of the strip for detecting IBV was 10-1.4522 EID50/mL. The strip showed a broad-spectrum cross-reactive capacity for detecting IBV antigens, including multiple IBV genotypes in China and all of the seven serotypes of IBV that are currently prevalent in southern China. Additionally, the result can be observed within 2 min without any equipment. The throat and cloacal swab samples of chickens that were artificially infected with three IBV strains were tested using the developed strip and the qPCR method; the strip test demonstrated a high consistency in detecting IBV via qPCR gene detection. In conclusion, the immunochromatographic strip that was established is rapid, sensitive, specific, simple, practical, and broad-spectrum; additionally, it has the potential to serve as an on-site rapid detection method of IBV and can facilitate the surveillance and control of the disease, especially in resource-limited areas.