PRRSV抗体竞争ELISA检测方法的建立与标准化研究  

Establishment and application of a competitive ELISA for detection of antibody against PRRSV

作　　者：夏向荣[1] 柏庆荣 柏坤桃[2] 黄灿平[1] 姜平[1] 

机构地区：[1]南京农业大学动物医学院农业部动物疫病诊断与免疫重点开放实验室,江苏南京210095 [2]江苏省高邮市畜牧兽医站,江苏高邮225600

出　　处：《畜牧与兽医》2005年第11期7-10,共4页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(30270990);浙江攻关项目(021102529)。

摘　　要：以猪繁殖与呼吸综合征病毒(PRRSV)核衣壳蛋白(N蛋白)基因的原核表达产物重组N蛋白为包被抗原,利用兔抗重组N蛋白血清和PRRS猪血清竞争该抗原,建立间接竞争ELISA方法检测PRRS抗体。经研究确定重组N蛋白的包被浓度为0·3μg/mL,检测血清不用稀释,兔抗重组N蛋白血清的工作浓度为1∶3000,酶标抗体的工作浓度为1∶10000,包被液为0·05mol/L、pH9·6的碳酸盐缓冲液。该方法特异性强,稳定性和重复性好,整个检测过程可在3h内完成。以IDEXX试剂盒的检测结果为参照标准,该方法的敏感性为79·76%,特异性为90·9%,符合率为83·59%。将该方法按试剂盒要求标准化,4℃放置5个月,检测效果不变。A competitive ELISA （eELISA） was developed based on recombinant nueleocapsid protein of porelne reproductive and respiratory syndrome virus （PRRSV） expressed by E. coll. Optimium conditions of the ELISA were developed as following： 0. 3 μg/mL reeombinant N protein in carbonate sodium buffer （0. 05 mol/L, pH 9. 6） for coating ELISA plates; 1：3 000 dilution of working titer for rabbit serum; 1 ： 10 000 dilution of working titer for HRP-labelled IgG; 〉 30% of inhibition rate as positive judging standard. Compared with IDEXX-ELISA, the sensitivity and specificity of the eELISA were 79. 76% and 90. 9%, respectively. The results indicated that the competitive ELISA was specific, sensitive and suitable for large scale sero-epidemiologieal studies for pigs infected with PRRSV. It is useful in diagnosis and surveillance of piglet＇s paraphoid.

关 键 词：PRRSV 抗体 诊断 竞争ELISA 

分 类 号：S858.28[农业科学—临床兽医学]