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The truncated spike protein (S1, 390-789aa) of porcine epidemic diarrhea virus was expressed as a recombinant protein in Baculorvirus expression system. Female BALB/c mice were immunized with the purified recombinant S1 (rS1) protein and two monoclonal antibodies, designated 8A3A10 and 12C4G12, were generated by hybridoma technique. The two monoclonal antibodies specifically reacted with PEDV rS1 protein, as proven by Western blot and indirect immunofluorescence assay. 8A3A10 monoclonal antibody showed a neutralizing activity to PEDV, as demonstrated in the results of fluorescent focus neutralization assay. These two newly established monoclonal antibodies may work as a useful tool to study the pathogenic mechanisms of PED virus and to develop diagnostic reagents.