van Nieuwstadt A P, Zetstra T
Department of Virology, Central Veterinary Institute, Lelystad, The Netherlands.
Am J Vet Res. 1991 Jul;52(7):1044-50.
A blocking ELISA was developed to detect antibodies directed against porcine epidemic diarrhea virus (PEDV). The PEDV antigen was first incubated with dilutions of test sera. Any antigen that was not blocked by antibodies in the serum was assayed in a double-antibody sandwich ELISA, using 2 monoclonal antibodies directed against different antigenic sites on PEDV as capture and detecting antibodies, respectively. The blocking ELISA was compared with a fixed-cell ELISA that used monolayers of Vero cells infected with PEDV prototype strain CV777 as a solid phase and a conjugate of an IgG-specific monoclonal antibody for antibody detection. Pigs were inoculated with PEDV strain CV777 or 1 of 2 field isolates, and antibody responses were measured by use of the 2 tests. Antibodies were detected by the blocking ELISA as early as postinoculation day 7 and, by the fixed-cell ELISA, as early as postinoculation day 14. From day 14 on, antibody titers for both tests correlated highly. Titers for the fixed-cell ELISA were 5.4 times higher than those for the blocking ELISA. The latter technique is easier to perform and discriminates well between infected and noninfected pigs, which makes this test useful for routine diagnosis and serologic surveys of porcine epidemic diarrhea.
开发了一种阻断ELISA法来检测针对猪流行性腹泻病毒（PEDV）的抗体。首先将PEDV抗原与待测血清的稀释液孵育。血清中未被抗体阻断的任何抗原，在双抗体夹心ELISA中进行检测，分别使用2种针对PEDV不同抗原位点的单克隆抗体作为捕获抗体和检测抗体。将阻断ELISA法与固定细胞ELISA法进行比较，后者使用感染PEDV原型株CV777的单层Vero细胞作为固相，并用IgG特异性单克隆抗体的缀合物进行抗体检测。给猪接种PEDV株CV777或2种田间分离株中的1种，并通过这2种检测方法测量抗体反应。阻断ELISA法早在接种后第7天就能检测到抗体，而固定细胞ELISA法最早在接种后第14天能检测到抗体。从第14天起，两种检测方法的抗体滴度高度相关。固定细胞ELISA法的滴度比阻断ELISA法高5.4倍。后一种技术操作更简便，能很好地区分感染猪和未感染猪，这使得该检测方法可用于猪流行性腹泻的常规诊断和血清学调查。
