摘要
用差速离心结合琼脂糖凝胶层析纯化的鸡传染性支气管炎病毒（ＩＢＶ）制备抗原免疫兔，获得高效价的多克隆免疫血清，提纯ＩｇＧ后用辣根过氧化物酶标记，建立了检测ＩＢＶ的斑点酶联免疫吸附试验（Ｄｏｔ－ＥＬＩＳＡ）方法。对影响本方法因素的研究表明，当酶标抗体作１：１００稀释、工作时间为３７℃、６０ｍｉｎ时结果最理想；对待检组织用氯仿处理、对硝酸纤维素膜用０．３％Ｈ２Ｏ２处理３０ｍｉｎ可有效地消除组织内过氧化物酶的影响。该方法具有较强的特异性和敏感性，对病毒的最低检出量为０．０１２４ｍｇ／ｍＬ。本法不仅可检测到纯化ＩＢＶ，而且可直接用于检测鸡胚尿囊液或病变组织（如气管、肾）中的病毒；对临床上疑似ＩＢＶ感染的病鸡的阳性检出率达７８．６％，而病毒分离率仅达６０％，两者符合率为９０％。试验结果表明，Ｄｏｔ－ＥＬＩＳＡ可作为ＩＢＶ早期诊断的方法，具有简单、快速、样品用量少等优点。
A dot enzymelinked immunosorbent assay (DotELISA)for avain infectious bronchitis virus (IBV)was developed.Rabbit antiIBV IgG obtained for the rabbit, which had been immuned by the purified IBV,and the IgG conjugated to horseradish-peroxidase(HRP),The best results showed tnat:the working concentration of the rabbit antiIBV IgG conjugated to HRP was 1:100,the incubation time was 60 minutes at 37℃;the virus was collected from the tissue by chloriform;and the filter paper had been incubated in 0.3%H2O2 for 30 minutes . The test is specific and sensitive,can detect the antigen more than 0.0124mg/ml.The test can find the IBV,not only of the purified virus but also in ammonialloic fluid and in the tissue (such as the pathotic bronchii and kidney ).the measured positive detection rate of the Dot-ELISA to IBV from the tissues of suspected IBV infected chickens was 78.6%,but the rate of isolation virus was only 60%,correlation between these two methods was 90%.The test is easy to run ,quickly and only need little antigen .
出处
《中国动物检疫》
CAS
1998年第3期4-7,共4页
China Animal Health Inspection
关键词
鸡
传染性支气管炎
病毒
检测
Infectious bronchitis virus Dot-ELISA Virus isolation
