IBV抗体的银加强金标免疫技术检测

Detection on Antibody to Infectious Bronchitis Virus of Chicken by Silver-Enhanced Colloidal Gold Assay (SECGA)

【研究目的】建立可在临床上检测鸡传染性支气管炎抗体的银加强金标免疫技术(SECGA);【方法】将胶体金标记纯化的兔抗鸡IgG,然后将IBV纯化抗原包被于NCM上(0.4ug/片),封闭后在膜片上滴加不同稀释度(10×2X)的血清,作用10分钟,洗涤后浸于1:4金标兔抗鸡IgG液中作用60分钟,再银染10分钟观察结果;【结果】SECGA能检出IB阳性血清的抗体>10×27,而不与ND、EDS76、IBD阳性血清发生有意义的交叉反应(抗体滴度<10×22)。对鸡IgG的最小检测量为0.88ng。用SECGA、气管环中和试验、ELISA试验检测IB抗体有明显的相关性;【结论】银加强金标免疫技术(SECGA)可用于IB抗体的检测,具有敏感、特异、简单、快速、适于现场诊断等优点,适宜于广大基层单位使用。

[Objective] A Silver-Enhanced Colloidal Gold Assay was developed for detection on anti-IBV antibody in chicken. [Method] The purified rabbit anti-chicken IgG was labeled with colloidal gold of 10nm in diameter. Then purified IBV was coated onto NCM with 0.4μg each dot. After being blocked, the NCM was immersed in serum samples diluted with different titer （10×2^x） for 10 minutes and then reacted in solution of rabbit anti-chicken IgG labeled with colloidal gold diluted with 1：4 for 1 hour. In the end NCM was stained 10 minutes by silver and the result was observed. [Results] IBV positive serum titer was more than 10×2^7, whereas ND, IBD, EDS-76 positive serum titer was less than 10×2^2, which indicated cross-reaction had no signification. The detection limit of chicken IgG was 0.88ng. SECGA, Serum Neutralization in TOCS and ELISA were significantly co-relative when anti-IBV antibody was detected using them respectively. [Conclusion] These results indicated that SECGA could be used for antibody surveillance of IB. At the same time the assay does not need specially experimental instruments, and the detected results are objective and apt to judge with the naked eye. Therefore the method is suitable for using widely in the basic farms and firms.