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J Vet Sci. 2018 Nov 26. [Epub ahead of print]

Novel application of Influenza A virus inoculated chorioallantoic membrane to characterize a nucleoprotein specific monoclonal antibody for immunohistochemistry assay.

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Department of Epidemiology, Animal Health Research Institute, Council of Agriculture, New Taipei City 25158, Taiwan.
Department of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, Taipei 10617, Taiwan.
Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan.


Monoclonal antibody (MAb) is widely applied in disease diagnosis. Here we report a MAb, 1 WF-4, against Influenza A virus nucleoprotein (NP), its broad response with Influenza A virus, and its application in immunohistochemistry (IHC) assay. WF-4 was screened by immunofluorescence assay (IFA). The results showed that its reactivity with baculovirus expressed full-length recombinant NP (rNP) in western blot (WB) and applicability in IHC. Fifteen Influenza A virus reference subtypes, H1 to H15, infected chicken embryonated chorioallantoic membranes (CAM) fixed by formalin, were all detectable in WF-4 based IHC. Also, the reactivity of the IHC test with NP from experimentally inoculated H6N1 and from all recent outbreak of H5 subtype avian Influenza A virus (AIV) field cases in Taiwan showed positive results. Our data indicate that CAM, the by-product of Influenza A virus preparation, is helpful for Influenza A virus specific MAb characterization, and that MAb WF-4 recognizes conserved and linear epitopes of Influenza A virus NP. Therefore, WF-4 is capable of detecting NP antigen in IHC and may be suitable for developing various tests for diagnosis of Influenza A virus, and especially AIV infection.


Influenza A virus; chorioallantoic membrane; immunohistochemistry; monoclonal antibody; nucleoprotein


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