Objective To prepare monoclonal antibodies against porcine epidemic diarrhea virus (PEDV) N protein, and develop an indirect immuno-fluorescence assay method used for detecting PEDV.
Method The expressed recombinantly PEDV N protein was used as an immunogen and 8-week-old female BALB/c mice were immunized. Then their spleen cells with high antibody titer were isolated and fused with SP2/0 cells. The hybridoma cell lines secreting monoclonal antibodies against PEDV N protein were screened. In Vero cells infected with PEDV, monoclonal antibody of anti-PEDV N protein was used as the primary antibody and FITC-goat-anti-mouse IgG was used as the secondary antibody to develop indirect immuno-fluorescence assay method used for detecting PEDV.
Result The prepared hybridoma cell lines could stably secrete anti-PEDV N protein antibodies, ELISA antibody titer in cell supernatant was above 1∶3 200, and in mouse ascites above 1∶1 000 000. While monoclonal antibodies were applied in established indirect immuno-fluorescence assay, the optimal conditions were that cells were fixed with 80% (φ) acetone at −20 ℃ for 30 min; The primary antibody was diluted 1 000 times by PBS buffer solution and incubated at 4 ℃ overnight; The secondary antibody was diluted 100 times by PBS buffer solution and incubated at 37 ℃ for 1 h. Transmissible gastroenteritis virus (TGEV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine reproductive virus (PRV), porcine enteric α corone virus (PEAV), porcine rotavirus (PoRV) and PEDV were detected by established indirect immuno-fluorescence assay method, only PEDV showed positive, all the else viruses showed negative.
Conclusion An anti-PEDV N protein monoclonal antibody is prepared, and the indirect immuno-fluorescence assay method used for detecting PEDV is established with high specificity. It provides an effective method for laboratory detection of PEDV and for localization and dynamic distribution of PEDV in infected cells.
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