间接ELISA筛选鹅源副粘病毒病单克隆抗体方法的建立  

Establishment of an Indirect ELISA for Detection of Monoclonal Antibody against Goose Paramyxovirus

作　　者：陈红英[1] 张书松[1] 邱蜜蜜[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002

出　　处：《河南农业大学学报》2008年第4期406-409,共4页Journal of Henan Agricultural University

基　　金：国家"十五"食品安全重大攻关专项(2001BA804A30-11)

摘　　要：将鹅源副粘病毒NA-1株接种SPF鸡胚进行病毒增殖,用蔗糖密度梯度离心对病毒进行纯化.用提纯的鹅源副粘病毒作为抗原包被ELISA板,通过反应条件的筛选,建立检测鹅源副粘病毒病单克隆抗体的间接ELISA方法.结果表明,抗原最佳包被质量浓度为5 mg.L-1,血清最适稀释度1∶80,其作用时间为60 min,羊抗鼠酶标二抗的最适质量浓度为1∶6 400,最适作用时间为60 min,37℃显色15 min.不与小鹅瘟等3种疫病阳性血清发生反应.对282孔杂交瘤细胞液进行检测,阳性检出率为12.77%,高于血凝抑制试验的阳性检出率(10.28%),符合率为91%.To establish indirect ELISA for detection of monoclonal antibody against goose paramyxovirus, goose paramyxovirus NA-1 isolate was inoculated into 10-day SPF chicken embryos, the allantoic fluid （AAF） was harvested. Goose paramyxovirus purified with sucrose gradient centrifugation was used to immunize mice and coat ELISA plate. The results showed the optimal concentration of coating antigen was 5 mg·L^-1. The serum sample was diluted in 1：80 and incubated for 60 min at 37 ℃. The dilution of conjugate was 1 ： 6 400, and the reaction time was 60 min. The OPD substrate was added and incubated at 37 ℃ for 15 min before terminated with stopping solution. The antigen had no cross reaction with antibodies to other 3 goose diseases. 36 of 282 wells of hybridoma ceils （ 12.77% ） were positive in indirect ELISA and 29 of 282 were positive in HI. The coincidence rate was 91% between indirect ELISA and HI test.

关 键 词：鹅源副粘病毒 抗体 间接ELISA 

分 类 号：S858.335[农业科学—临床兽医学]