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.
出处
《中国兽医学报》
CAS
CSCD
北大核心
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)
关键词
猪繁殖与呼吸综合征病毒
ELISA
重组N蛋白
porcine reproductive and respiratory syndrome virus
ELISA
recombinant nucleoprotein
