重组N蛋白抗原检测PRRSV抗体ELISA的研究 Ⅰ.ELISA方法的初步建立及其标化  

Development of ELISA Kit Using Recombinant Nucleoprotein as Antigen of PRRS Virus Ⅰ.The Optimal Reaction Conditions of ELISA

作　　者：吴延功[1] 徐天刚[1] 王志亮[1] 张喜悦[1] 王君玮[1] 宋翠平[1] 刘佩兰[1] 徐培莲[1] 宋芳[1] 许立华[2] 陈溥言[2] 

机构地区：[1]农业部动物检疫所,山东青岛266032 [2]南京农业大学动物医学院,江苏南京200095

出　　处：《中国兽医学报》2005年第4期339-342,345,共5页Chinese Journal of Veterinary Science

基　　金：国家"十五"重大专项科技攻关项目(2002BA514A-18);农业科技成果转化资金项目(02EFN213710657)

摘　　要：以纯化的重组N蛋白作为包被抗原,对各种条件进行优化(如抗原的包被,作用时间及底物的选择),建立了检测猪繁殖与呼吸综合征病毒(PRRSV)抗体的间接ELISA方法。研究结果表明,重组抗原最适包被质量浓度为1mg/L,最适包被条件为4℃过夜,血清稀释度为1∶40,待检血清和酶标二抗的反应时间分别为37℃30min和40min,底物用TMB溶液,37℃显色10min。本研究对ELISA板的蛋白稳定剂、血清稀释液的显色系统和终止液进行了研究。用全病毒进行的阻断试验结果表明,全病毒能够阻断阳性血清与包被重组N蛋白抗原的结合反应。用此方法对几种繁殖障碍性疾病的阳性血清进行检测,均为阴性,无交叉反应,表明建立的ELISA具有很好的特异性。板内和板间重复性试验结果为,同1份血清板内各孔的变异系数和板间各次的变异系数均小于7%,表明本方法的重复性好。The purified N protein was used to be coated on the well of 96-well plate, each following step was optimized. As a result, an indirect ELISA was constructed to detect antibody against PRRSV. The various factors and conditions of ELISA were explored, and the optimal reaction conditions of ELISA were determined. It was shown that the optimal concentration of recombinant N protein for coating of plate was1 mg/L, the optimal coating condition of recombinant N protein for ELISA was 4℃ overnight, the dilution of serum sample was 1∶40, serum sample for detecting and HRP-labeled rabbit anti-porcine IgG should be incubated at 37℃ for 30 min and 40 min respectively, the substrate for ELISA was incubated at 37℃ for 10 min before terminated with the stopping solution.The blocking test for the reaction between the PRRSV positive serum and the recombinant N protein was carried out by using the PRRSV antigen.The results showed that PRRS virus could block the reaction completely. Meanwhile, it was confirmed that there was no cross reaction between the antibodies against other porcine infectious diseases.The results revealed that the indirect ELISA by the purified recombinant nucleoprotein had good specificity for the detection of PRRSV antibody in serum. The difference value among wells in a plate and among plates for ELISA was both less than 7%, which showed the assay had a good retrievality.

关 键 词：猪繁殖与呼吸综合征病毒 ELISA 重组N蛋白 

分 类 号：S852.65[农业科学—基础兽医学]