J Vet Sci.  2019 Jan;20(1):51-57. 10.4142/jvs.2019.20.1.51.

Novel application of Influenza A virus-inoculated chorioallantoic membrane to characterize a NP-specific monoclonal antibody for immunohistochemistry assaying

  • 1Department of Epidemiology, Animal Health Research Institute, Council of Agriculture, New Taipei City 25158, Taiwan.
  • 2Department of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, Taipei 10617, Taiwan. ivancheng@ntu.edu.tw
  • 3Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan.


Monoclonal antibodies (MAbs) are widely applied in disease diagnoses. Herein, we report a MAb, WF-4, against Influenza A virus nucleoprotein (NP), its broad response with Influenza A virus, and its application in an 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), indicating its IHC applicability. Fifteen Influenza A virus (reference subtypes H1 to H15) infected chicken embryonated chorioallantoic membranes (CAM), fixed by formalin, were all detectable in the WF-4-based IHC assay. Also, the reactivity of the IHC test with NP from experimentally inoculated H6N1 and from all recent outbreaks of H5 subtype avian Influenza A virus (AIV) field cases in Taiwan showed positive results. Our data indicate that CAM, a by-product of Influenza A virus preparation, is helpful for Influenza A virus-specific MAb characterization, and that the WF-4 MAb recognizes conserved and linear epitopes of Influenza A virus NP. Therefore, WF-4 is capable of detecting NP antigens via IHC and may be suitable for developing various tests for diagnosis of Influenza A virus and, especially, AIV infection.


Chorioallantoic membrane; Immunohistochemistry; Influenza A virus; Antibodies, Monoclonal; Nucleoproteins

MeSH Terms

Antibodies, Monoclonal
Blotting, Western
Chorioallantoic Membrane*
Disease Outbreaks
Fluorescent Antibody Technique
Influenza A virus
Influenza in Birds
Influenza, Human*
Antibodies, Monoclonal


  • Fig. 1 Western blot analysis of monoclonal antibody WF-4. (A) Lane 1, molecular weight standard; Lane 2, H5N2/1209; Lane 3, baculovirus-expressed recombinant nucleoprotein. (B) Lane 1, molecular weight standard; Lane 2, A/wild duck/Tainan/1634/09 (H1N1); Lane 3, H5N2/1209; Lane 4, A/duck/Tainan/A30/02 (H5N2); Lane 5, A/wild duck/830/05 (H5N2); Lane 6, A/duck/Hong Kong/820/80 (H5N3); Lane 7, A/chicken/Miaoli/2904/00 (H6N1); Lane 8, A/duck/Tainan/A45/03 (H7N7).

  • Fig. 2 Twelve-day-old chicken embryo. (A) Normal chorioallantoic membrane (CAM). (B) Negative control for immunohistochemistry (IHC) assay. CAM infected by avian Influenza A virus reference subtypes and collected at 2 days post-inoculation. (C) H11N6. (D) H11N6. (E) H14N5. (F) H15N8. Ec, fibrous chorionic ectoderm; M, mesoderm; En, allantoic endoderm. H&E stain (A). IHC stain (B–F). Scale bars = 20 µm (D–F), 100 µm (C), 200 µm (A and B).

  • Fig. 3 Immunohistochemistry (IHC)- and H&E-stained tissue samples of experimentally infected chicken and poultry field cases. (A–C) Kidney from H6N1 experimentally infected via the intranasal route, clinically normal chicken at 5 days post-inoculation. (A) Multifocal IHC-positive signals densely distributed in the renal parenchyma. (B) Scattered degeneration, necrosis, and regeneration of renal tubular epithelium were observed in this H&E-stained kidney section. Spheroids of material clusters and calcification in the lumen of necrotic tubules were common. (C) Renal necrosis at different stages. (D) H5N2 highly pathogenic avian Influenza A virus (HPAIV) field cases in 2012. Lymphocytic laryngitis with necrosis of mucous gland cells (arrow). Avian Influenza A virus (AIV) nucleoprotein (NP) in individual epithelium. (E) Naturally infected with HPAIV H5N8 in goose, the influenza virus antigen is noted in the cardiac fibers. (F) Focal necrosis surrounded by hepatocytes with intranuclear and intracytoplasmic staining for AIV NP in chicken naturally infected with HPAIV H5N2 virus. The NP was immunolabeled at high levels in the endothelial cell lining of liver. (G) AIV antigens were identified in the primordial follicles and lymphocytes of the ovarian cortex. (H) There was specific immunolabeling in the nucleus and cytoplasm of neurons, glial cells, axons, and endothelial cells in duck naturally infected with HPAIV H5N6 virus. IHC stain (A, C–H). H&E stain (B). Scale bars = 20 µm (D and F), 50 µm (B, C, G, and H), 100 µm (E), 200 µm (A). a, normal tubular epithelium; b, pyknosis of tubular epithelial cells; c, karyorrhexis and heterophil infiltration; d, formation of urate tophi; e, normal; f, degeneration with minute IHC-positive staining in nucleus; g, necrotizing tubular cells with moderate IHC signals in both nucleus and cytoplasm; h, tubular cells with pyknosis covered by strong positive signals.


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