Preparation and Identification of Neutralizing Monoclonal Antibodies against Egg Drop Syndrome Virus and the Use in Sandwich ELISA
Recombinant egg drop syndrome virus(EDSV)fiber protein was obtained and its hemagglutination(HA)activity was identified in order to generate neutralizing monoclonal antibodies against EDSV.The HA titer of the purified fiber protein reached 1∶27.The recombinant fiber protein was used as an immunogen to immunize BALB/c mice.The indirect enzyme-linked immunosorbent assay(iELISA)and indirect immunofluorescence assay(IFA)titers of the mice serum were up to 1∶409600 and 1∶12800,respectively,after four immunizations.Hybridoma cells were prepared by cell fusion using spleen cells from immunized mice.Two hybridoma cell lines,9G12 and 10E11,which could secrete neutralizing monoclonal antibodies stably,were effectively developed after subcloning and screening with iELISA and IFA.The ascites titers of monoclonal antibodies 9G12 and 10E11 detected by iELISA were 1∶128000 and 1∶1020000,respectively.The titers of IFA were 1∶2000 and 1∶8000,respectively.Both monoclonal antibodies were unable to react selectively with the denatured EDSV fiber protein,according to the Western blot data.Viral neutralization test confirmed that 9G12 and 10E11 had neutralization activity.The neutralization titers of 9G12 and 10E11 were 1∶27 and 1∶24,respectively.The heavy chains of the antibodies were IgG1 subtype,whereas the light chains were Kappa type.A sandwich ELISA method based on monoclonal antibodies 9G12 and 10E11 was applied to the detection of EDSV in clinical samples,and the coincidence rate with real-time PCR results was 91.7%.In conclusion,two hybridoma cell lines,9G12 and 10E11,which secreted specific anti-EDSV antibodies,were successfully screened in this study.Both monoclonal antibodies had the activity of neutralizing EDSV infection and could be applied to clinical detection of EDSV.
Egg drop syndrome virus(EDSV)Fiber proteinNeutralizing monoclonal antibodiesAntibody neutralization titerSandwich ELISA
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维普
