检测传染性支气管炎抗体NP-ELISA方法的建立

【研究目的】建立可在临床上检测鸡传染性支气管炎抗体的检测IBV抗体的间接ELISA方法；【方法】以表达的重组IBV NP 蛋白为包被抗原，将之包被于聚苯乙烯酶标板上，以封闭液进行封闭，然后加待检血清在37℃作用1h，洗涤后加酶标兔抗鸡IgG抗体，在37℃作用1h，加底物显色，终止反应后测定OD值；【结果】抗原包被浓度为1.45mg/ml，待检血清最佳稀释度为1:40，酶标二抗最佳工作浓度为1:1000，确定阳性判定标准为OD450≥0.314。NP-ELISA与AIV H9、H5、H7、IBD、ND、EDS76 的标准阳性血清不发生交叉反应，具有良好的特异性；【结论】NP-ELISA具有特异性强、灵敏度高、重复性好、方便快速的特点,可用于临床IB抗体的检测。

Development of NP-ELISA for Detection of Antibody against Infectious Bronchitis Virus

【Objective】A nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus.【Method】The purified recombinant IBV nucleoprotein was coated onto the Polystyrene plate and sealed up with confining liquid. Then the serum sample was added on it and incubated at 37℃ for 1 hour. When the plate was washed, the rabbit anti-chicken IgG labeled with HRP was put onto it for being incubated at 37℃ for 1 hour too. And then the substrate was added for coloring. OD value was assayed after the reaction being ended.【Results】The optimal concentration of coating antigen was 1.45μg/ml, and the optimal dilution of serum and rabbit anti-chicken IgG labeled with HRP were 1:40 and 1:1000 respectively. The detection-limit between negative and positive serum in OD450 value was 0.310, when the OD450 value was equal or over 0.314, it was positive, or else negative. NP-ELISA had no reaction to the positive serums of AIV H9、H5、H7、ND、IBD and EDS76 , which directed that the expressed NP protein was only reacted to the serum of IBV. 【Conclusion】The results above indicated the NP-ELISA afforded strong specificity, high sensitivity , excellent coincidence and could be used for antibody surveillance of IB in clinical practice.