摘要
【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IBVNP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1.45μg/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AIVH9、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。
[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.
出处
《中国农学通报》
CSCD
2007年第8期48-52,共5页
Chinese Agricultural Science Bulletin
