摘要
[Objective] The aim of this study was to develop an indirect sandwich EUSA method for detection of swine vesicular disease virus （SVDV）. [Method] High titer serum against SVDV was prepared respectively by inoculated rabbits and guinea pigs with purified virus. To develop an indirect sandwich ELISA, the optimum concentrations of capture antibody, detection antibody, enzyme conjugate and standard antigen were determined using block titration, and positive threshold value was also determined. The specificity, sensitivity and repeatability of the developed IELISA were evaluated using cross-reaction test, comparison test and intra-assay repeated test. In addition, standard samples and clinical samples were detected by this method. [ Result] The best working conditions of the developed ELISA are as follows： capture antibody, 1：400; detection antibody, 1 ： 200; enzyme conjugate, 1 ： 8 000; and standard antigen, 1 ： 4. The positive threshold value was found to be 0.20. For the detection by the developed EUSA, no cross-reaction with foot and mouth disease was observed. The developed ELISA had close sensitivity with ELISA recommended by the World Organisation for Animal Health （OIE） but had sensitivity 2 -4 times higher than that of reverse indirect hernagglutination test. In addition, the developed method also had good reproducibility, and the detection results of standard samples were in line withtheir own background. All the 36 clinical samples were negative in the developed ELISA. [ Conclusion] The developed indirect sandwich ELISA can be used for diagnosis of swine vesicular disease.
[Objective] The aim of this study was to develop an indirect sandwich EUSA method for detection of swine vesicular disease virus （SVDV）. [Method] High titer serum against SVDV was prepared respectively by inoculated rabbits and guinea pigs with purified virus. To develop an indirect sandwich ELISA, the optimum concentrations of capture antibody, detection antibody, enzyme conjugate and standard antigen were determined using block titration, and positive threshold value was also determined. The specificity, sensitivity and repeatability of the developed IELISA were evaluated using cross-reaction test, comparison test and intra-assay repeated test. In addition, standard samples and clinical samples were detected by this method. [ Result] The best working conditions of the developed ELISA are as follows： capture antibody, 1：400; detection antibody, 1 ： 200; enzyme conjugate, 1 ： 8 000; and standard antigen, 1 ： 4. The positive threshold value was found to be 0.20. For the detection by the developed EUSA, no cross-reaction with foot and mouth disease was observed. The developed ELISA had close sensitivity with ELISA recommended by the World Organisation for Animal Health （OIE） but had sensitivity 2 -4 times higher than that of reverse indirect hernagglutination test. In addition, the developed method also had good reproducibility, and the detection results of standard samples were in line withtheir own background. All the 36 clinical samples were negative in the developed ELISA. [ Conclusion] The developed indirect sandwich ELISA can be used for diagnosis of swine vesicular disease.
基金
funded by the Major Sci-Tech Fund of Gansu Province (092NKDA032)
