牛病毒性腹泻病毒夹心ELISA快速检测方法的建立与初步应用

Establishment and preliminary application of the sandwich ELISA method for quick detection of bovine viral diarrhea virus

用增殖与浓缩后的牛病毒性腹泻病毒(BVDV)分别免疫健康牛犊和家兔,制备牛抗BVDV和兔抗BVDV超免疫血清,并用层析法纯化牛抗BVDV IgG和兔抗BVDV IgG,建立了检测BVDV的双抗体夹心ELISA快速检测方法.结果表明:该检测方法的最佳反应条件为:牛抗BVDV IgG包被浓度为100μg/mL,兔抗BVDV IgG最佳工作浓度为50μg/mL,样品反应时间为90min,酶标抗体工作浓度为1∶8 000,以D490nm≥0.177作为阳性判定标准.该ELISA检测方法的重复性CV小于10%,最低检测限为1.87μg/mL,与牛轮状病毒、牛冠状病毒、猪瘟病毒、犊牛大肠杆菌和沙门菌等病原无交叉反应,该ELISA试剂在室温下至少可保存6个月.用该ELISA方法和IDEXX公司抗原检测试剂盒对甘肃省不同地区牛场156份临床粪便样品进行了检测,阳性检出率分别为17.3%和16.7%.表明本试验建立的ELISA方法特异性强、敏感性高且重复性好,可用于BVDV的快速检测.

Two kinds of hyperimmune sera, bovine anti-BVDV IgG and rabbit anti-BVDV IgG, were prepared by inoculating respectively calves and rabbits with bovine viral diarrhea virus （BVDV） purified by proliferation and concentration,and purified by affinity chromatography. Based on the two kinds of IgG, a sandwich ELISA for quick detection of BVDV was established. The results showed that the optimal coating concentration of bovine anti-BVDV IgG was 100 ffg/mL,the optimal working concentration of rabbit anti- BVDV IgG was 50 ffg/mL, the reaction time of sample was 90 min, and the optimal working dilution of HRP-labelled sheep anti-rabbit IgG was 1 = 8 000. The positive standard value was more than 0. 177 （D490）. The coefficient of variation of reproducibility was less than 10%, and at least antigen limitation for detec- tion was 1.87μg/mL. The ELISA had no cross-reaction with bovine rotavirus, bovine coronavirus, classical swine fever virus,Escherichia coli and Salmonella. The prepared plates and reagents could preserved at least six months at room temperature. 156 fecal samples from the fields of cattle farm of Gansu Provincewere detected by the ELISA and detection kit producted by 16.7 % respectively. The results revealed that the ELISA and high sensitivity,indicating a suitable method for quick 3 % and ucibility, IDEXX, and the positive ratio were 17. possessed good specificity and reprod detection of BVDV.