Preparation and Identification of Monoclonal Antibodies Distinguishing Porcine epidemic diarrhea virus Variants from Classical Strains
WANG Ya-Wen1,*, ZHAO Xue1,*, LI Tan-Qing1, MI Jian-Hua2, MIAO Yun-Yan2, SUN Li-Dan1, ZHANG Yi-Ming1, SONG Qin-Ye1,*
1 Research Center of Veterinary Biologicals Engineering and Technology of Hebei/College of Veterinary Medicine, Hebei Agricultural University, Baoding 071000, China; 2 Hebei Laishui County Animal Husbandry and Aquaculture Bureau, Baoding 074199, China
Abstract：Porcine epidemic diarrhea (PED) caused by Porcine epidemic diarrhea virus (PEDV) is a highly contagious and devastating enteric infectious disease to pig (Sus scrofa) production. Serological techniques are absent for distinguishing PEDV variants from classical strains. Monoclonal antibody (McAb) is a kind of important and basic experimental materials for serological testing or diagnosing technique researches. In order to prepare McAb for distinguishing PEDV variants from classical strains, this study compared and analyzed the nucleotide and amino acid sequences of spike (S) protein of PEDV virulent variants and classical strains by the software of DNAMAN and DANStar7.1. The polypeptide, 55IGENQGVNSTWYCAGRHPTAS75 being different between the PEDV variants and the classical ones, was screened out and synthesized. BALB/c mice (Mus musculus) were immunized 3 times at an interval of 2 weeks with the polypeptide conjugated keyhole limpet haemocyanin (KLH), and then one strain of hybridoma (named 3#) which secreted IgG2bκ monoclonal antibody (named McAb 3#) was obtained through cell fusion technique, 3 rounds of clone selecting as well as enzyme-linked immunosorbent assay (ELISA) detection. The McAb could specifically react with PEDV-S protein whereas it did not combine with PEDV nucleocapsid (N) protein by ELISA and Western blot, meanwhile, PEDV variants could specifically bind to the McAb 3# in Vero-81 cells using the indirect immunofluorescence assay (IFA). The specific ELISA antibody titer reached to 1∶106 in mouse ascitic fluid induced by 3# hybridoma cells. Moreover, IFA based on the McAb 3# was performed in antigen detection of PEDV variant HBQY2016 and the classical attenuated strain CV777 in Vero-81 cells, respectively. Green fluorescence signals were observed only in the cytoplasm of Vero-81 cells infected with PEDV variant HBQY2016, but not in the cells infected with the classical attenuated strain CV777, which indicated the McAb 3# could distinguish the PEDV variant from the classical strain. The results showed that the McAb prepared in this study specifically combined with PEDV S protein and could distinguish the PEDV variant from classical strain. Its preparation could be used for studying PEDV infection immunity and developing antigen or antibody detection methods to differentiate PEDV variants from classical strains.
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