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Generation, identification, and functional analysis of monoclonal antibodies against porcine epidemic diarrhea virus nucleocapsid.

ABSTRACT: The variant strains of porcine epidemic diarrhea virus (PEDV) severely threaten the pig industry worldwide and cause up to 100% mortality in suckling piglets. It is critically important and urgent to develop tools for detection of PEDV infection. In this study, we developed six monoclonal antibodies (mAbs) targeting N protein of PEDV and analyzed their applications on enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), western blot assay, and flow cytometry assay. The results demonstrated that all these six mAbs were IgG1 isotype and ? chain. Among these six mAbs, 3F12 recognizes a linear epitope (VAAVKDALKSLGI) while the other five mAbs recognize different conformational epitopes formed by a specific peptide fragment or the full length of N protein. The functional analysis showed that all these six mAbs were applicable to ELISA, western blot, IFA, and flow cytometry assay. In conclusion, we developed six mAbs against PEDV-N protein to facilitate the early detection of PEDV infection using ELISA, western blot, IFA, and flow cytometry.

SUBMITTER: Yang W

PROVIDER: S-EPMC7079923 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Generation, identification, and functional analysis of monoclonal antibodies against porcine epidemic diarrhea virus nucleocapsid.

Yang Wenting W Chen Wenwen W Huang Jingling J Jin Li L Zhou Yawei Y Chen Jianing J Zhang Na N Wu Donglai D Sun Encheng E Liu Guangliang G

Applied microbiology and biotechnology 20190315 9

The variant strains of porcine epidemic diarrhea virus (PEDV) severely threaten the pig industry worldwide and cause up to 100% mortality in suckling piglets. It is critically important and urgent to develop tools for detection of PEDV infection. In this study, we developed six monoclonal antibodies (mAbs) targeting N protein of PEDV and analyzed their applications on enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), western blot assay, and flow cytometry assay. ...[more]

PMID: 30877355

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Project description:Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea, which can cause death in suckling piglets. Vaccines confer only partial protection against new mutant strains, whereas antibodies targeting virus-encoded proteins may be effective prophylactics. In this study, we constructed a recombinant single chain variable fragment (scFv) library from the spleens of two pigs immunized with a recombinant PEDV nucleocapsid (N) protein. Among the positive clones directed against PEDV N protein isolated from the library, four scFvs that showed higher affinity for N were functionally analyzed. These scFvs specifically bound to the PEDV N protein, but not to the transmissible gastroenteritis virus (TGEV) N protein. Their framework regions were highly conserved, whereas their complementarity-determining regions displayed clear diversity. An immunofluorescence assay showed the co-localization of the four scFvs with PEDV N protein in cells. They significantly suppressed PEDV replication, detected with reverse transcription (RT)-quantitative PCR (qPCR; P < 0.01). Two of them significantly reduced the viral titer at 48 hpi and 72 hpi (P < 0.05). In addition, they observably suppressed the production of viral protein at 72 hpi. The expression of interferons, interferon regulatory factor 3 (IRF3), and IRF7 was assessed with RT-qPCR, which indicated that PEDV dramatically suppressed the transcription of interferon-λ1 and IRF7 and that the scFvs significantly upregulated their expression (P < 0.05). These findings facilitated the investigation of the mechanism by which PEDV evaded the host immune response and suggested that these porcine scFvs were potential candidate agents for the prevention and treatment of porcine diarrhea caused by PEDV. KEY POINTS: • Four scFvs targeting PEDV N protein were generated from porcine spleens • These scFvs co-localized with PEDV N protein and suppressed PEDV replication • These scFvs significantly upregulated IFN-λ1 expression.

Dataset Information

Generation, identification, and functional analysis of monoclonal antibodies against porcine epidemic diarrhea virus nucleocapsid.

Publications

Generation, identification, and functional analysis of monoclonal antibodies against porcine epidemic diarrhea virus nucleocapsid.

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