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

• Tu YC
• Chen KY
• Chen CK
• Cheng MC
• Lee SH
• Cheng IC

• KMID: 2434777
• DOI: http://doi.org/10.4142/jvs.2019.20.1.51

Abstract

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.

Keyword

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

MeSH Terms

Animals
Antibodies, Monoclonal
Blotting, Western
Chickens
Chorioallantoic Membrane*
Diagnosis
Disease Outbreaks
Epitopes
Fluorescent Antibody Technique
Formaldehyde
Immunohistochemistry*
Influenza A virus
Influenza in Birds
Influenza, Human*
Nucleoproteins
Taiwan
Antibodies, Monoclonal
Epitopes
Formaldehyde
Nucleoproteins

Figure

• 

  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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