Preparation And Characterization Of Monoclonal Antibodies Against Porcine Epidemic Diarrhea Virus,and Establishment Of ELISA For SIgA Antibody Detection

Porcine epidemic diarrhea virus（PEDV）,a member of the Alphacoronavirus genus within the Coronaviridae family,is a critical infectious disease pathogen endangering the swine industry.PEDV infection results in porcine epidemic diarrhea（PED）,which is characterized by acute diarrhea,vomiting,dehydration,and high mortality in neonatal piglets.Since 2010,highly virulent PEDV variants have emerged in swine farms in Asia,North America and Europe,leading to significant economic losses to the global swine industry.Passive lactogenic immunity remains a promising and effective way to protect neonatal suckling piglets from enteric diseases like PED.High titers of secretory Ig A（sIgA）antibody in milk will decrease morbidity and mortality in neonatal suckling piglets.In this study,monoclonal antibodies（MAbs）against PEDV were prepared and identified by various methods.Furthermore,an enzyme-linked immunosorbent assay（ELISA）for detecting anti-PEDV sIgA antibody in milk was established based on the viral antigen capture by the specific MAb.（1）Preparation of anti-PEDV MAbsIn order to prepare MAbs against PEDV,tangential flow system and gel filtration chromatography were applied to purify epidemic PEDV CH/hubei/2016 virions,which were utilized as immunogen to immunize the 6 to 8-week BALB/c mice according to conventional procedures.The spleen cells in the mouse with higher serum antibody titers were fused with SP2/0 myeloma.The hybridoma cell lines were screened by immunoperoxidase monolayer assay（IPMA）,and the positive ones were subcloned to obtain anti-PEDV MAbs.A total of10 MAbs against PEDV were obtained in this study.（2）Characterization of anti-PEDV MAbsIn this study,the structural protein expression vectors of PEDV were constructed.The MAbs were characterized by ELISA and Western-blot using PEDV recombinant structural proteins.Reactivity of the MAbs were further characterized by indirect immunofluorescence assay（IFA）and confocal laser scanning microscopy（CLSM）.The results showed that a total of four anti-PEDV spike protein（S）MAbs and six anti-PEDV nucleocapsid protein（N）MAbs were obtained.Ascites were prepared via in vivo induction in mice,and their IPMA titers were from 2.56×10^-4 to 1.024×10^-5.IFA showed that four MAbs were reacted with PEDV-infected Vero cells.In addition,CLSM showed the subcellular localization of newly synthesized S protein during PEDV infection in Vero cells using the anti-PEDV S protein MAb.（3）Establishment of ELISA for PEDV sIgA antibody detectionIn this study,Anti-PEDV S protein MAbs were utilized to capture PEDV antigens,and an ELISA was established to detect sIgA antibody against PEDV in clinical milk samples.The coefficient of variation of the repetitive tests was between 4.0%and 8.1%,showing good repeatability.When diluted 1280-fold,the clinical positive samples were still detected,showing high sensitivity.Clinical samples were simultaneously detected by the established ELISA and a commercial ELISA kit,with a high coincidence rate.In summary,this study successfully prepared and characterized 10 anti-PEDV MAbs,which provided an important molecular basis for research on PEDV infection mechanism and diagnostic methods.Furthermore,ELISA for detecting PEDV sIgA antibody was established based on viral antigen capture by anti-S MAb,which provided a powerful tool for evaluating vaccination efficiency against PEDV.