摘要
以纯化的原核表达的猪圆环病毒2型(PCV-2)重组结构蛋白(rCap蛋白)为包被抗原,建立PCV-2间接ELISA诊断方法;对抗原最适包被浓度、待检血清稀释浓度、重组抗原包被条件、封闭液的筛选、阴阳性临界值等条件进行了优化;对优化后的ELISA检测方法进行特异性试验、临床应用试验;组装试剂盒并进行试剂盒保存期试验。优化反应条件后确定的抗原最适包被浓度为2.815μg/mL,抗原最佳包被条件为37℃2 h;血清最适稀释度为1∶40,酶标抗体最适稀释度为1∶500,最佳封闭液为1%BSA;阴阳性临界值判定标准为D450 nm≥0.33,判为阳性,D450 nm<0.33,判为阴性。特异性试验结果表明,只有PCV-2阳性血清反应后的D450 nm>0.33,包括抗PCV-1阳性血清在内的其他5种抗血清反应后的D450 nm<0.33,说明所建立的ELISA方法具有良好的特异性。经对临床疑似PCV-2感染的100份血清样品进行检测,阳性检出率为69%。组装试剂盒在4℃条件下至少可保存12个月以上,说明所建立的诊断方法可以在生产中大量推广应用。本研究成功建立了能特异性检测抗PCV-2血清抗体的ELISA检测方法。
An indirect enzyme-linked immunosorbent assay （ELISA） method was established to detect antibodies against porcine circovirus type 2 using the recombinant capsid protein （rCap） expressed by prokaryotic pQE30 vector as coating antigen. The various factors affected the experiments such as concentration and coating time of rCap, the serum dilution concentration, etc were optimized, and the storage period of the ELISA kit was also tested. The results demonstrated that optimal concentration of rCap for coating was 2. 815 ℃g/mL, the optimal coating condition of rCap for ELISA was incubated at 37℃ for 2 hours, the dilution of serum sample was 1 -40, the work concentration of HRP-labeled rabbit anti-porcine IgG was 1 - 500, the best blocking buffer was BAS with 1 - concentration, and the critical value of positive and negative of ELISA was D450 nm= 0.33. The specificity test showed that the established ELISA method was only displayed D450nm〉0.33 for standard positive serum of PCV-2, but D450 nm〈0. 33 for other serums of PCV-1, CSFV, JEV, PRV, PRRSV, etc which acting as the negative controls. Sixty-nine of 100 clinic suspicious PCV-2 infection samples was positive by using the established ELISA method. The storage period test of the ELISA kit showed that the kit could be stable for at least 12 months at 4℃. This study indicated that an indirect ELISA method for detection PCV-2 in serum was successfully established, and could be used for the rapid clinic diagnosis of PCV 2.
出处
《中国畜牧兽医》
CAS
北大核心
2009年第5期53-57,共5页
China Animal Husbandry & Veterinary Medicine
基金
河南省科技攻关项目(0624410098)
