摘要
为了对鸡传染性支气管炎(IB)疫苗的免疫效果进行有效评估,研究以QX型IBV的Vero细胞适应株为包被抗原,对检测条件进行优化,建立了IBV抗体间接ELISA检测方法。结果显示:抗原最佳包被浓度为160μg/mL,最佳包被条件为37℃、2 h,封闭条件为5%脱脂奶粉、37℃封闭2 h,最佳抗体稀释液选择2%脱脂奶,一抗的最佳反应条件为37℃、60 min,二抗的最佳稀释度为1∶10000,作用时间为60 min,底物显色时间为15 min;特异性、重复性试验证明,该方法与其他病原无交叉反应,特异性好,批内、批间变异系数较低,重复性好。研究初步建立了IBV抗体间接ELISA检测方法,可应用于免疫鸡群后抗体水平监测及早期感染预警。
In order to effectively evaluate the immune effect of chicken infectious bronchitis(IB)vaccine,Vero cells of QX type IBV strain were used as coated antigen to optimize various detection conditions,and the indirect ELISA detection method of IBV antibody was established.The optimized reaction conditions were as follows:Optimal antigen coating concentration was 160μg/mL,the optimum coating conditions was 37℃for 2 h,the blocking solution was 5%non-fat skimmed milk,incubated at 37℃for 2 h,the best antibody diluent was 2%non-fat skim milk.Serum sample dilution was coated at 37℃for 60 min.The secondary antibody was diluted at 1∶10000 and incubated at 37℃for 60 min.Color development was performed with the TMB substrate and incubated at 37℃for 15 min.The specificity and repeatability tests showed that the method had no cross reaction with other pathogens and low intra-batch and inter-batch coefficient of variation.In this study,an indirect ELISA kit for IBV antibody was successfully established,it could be used for antibody level monitoring and early infection warning after immunizing chickens.
作者
程旭
周生
唐梦君
高明燕
俞燕
姜逸
CHENG Xu;ZHOU Sheng;TANG Mengjun;GAO Mingyan;YU Yan;JIANG Yi(Institute of poultry,Chinese Academy of Agricultural Sciences,Jiangsu,Yangzhou 225125;Qingdao Agricultural University,Shandong,Qingdao 266109;College of Veterinary Medicine Yangzhou University,Jiangsu,Yangzhou 225009)
出处
《中国家禽》
北大核心
2022年第5期25-31,共7页
China Poultry
基金
扬州市科技计划(现代农业)项目(YZ2020052)
江苏省自然科学基金面上项目(BK20201483)
江苏高校动物重要疫病与人兽共患病防控协同创新中心开放课题(R2014)
国家自然科学基金项目(31602091、31572524)
