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. 1999 May;6(3):341-4.
doi: 10.1128/CDLI.6.3.341-344.1999.

Production, characterization, and uses of monoclonal antibodies against recombinant nucleoprotein of elk coronavirus

G C Daginakatte  1 , C Chard-Bergstrom, G A Andrews, S Kapil
Affiliations

Production, characterization, and uses of monoclonal antibodies against recombinant nucleoprotein of elk coronavirus

G C Daginakatte et al. Clin Diagn Lab Immunol. 1999 May.

Abstract

This is the first report of the production of monoclonal antibodies against elk coronavirus. The nucleoprotein gene of elk coronavirus was amplified by PCR and was cloned and expressed in a prokaryotic expression vector. Recombinant nucleocapsid protein was used to immunize mice for the production of hybridomas. Twelve hybridomas that produced monoclonal antibodies against the nucleocapsid protein of elk coronavirus were selected by an indirect fluorescent-antibody test, an enzyme-linked immunosorbent assay, and a Western blot assay. Ten of the monoclonal antibodies were of the immunoglobulin G1 (IgG1) isotype, one was IgG2a, and one was IgM. All had kappa light chains. By immunohistochemistry four monoclonal antibodies detected bovine coronavirus and elk coronavirus in formalin-fixed intestinal tissues. Antinucleoprotein monoclonal antibodies were found to be better at ruminant coronavirus detection than the anti-spike protein monoclonal antibodies. Because nucleoprotein is a more abundant antigen than spike protein in infected cells, this was not an unexpected finding.

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Figures

FIG. 1
FIG. 1
SDS-polyacrylamide gel electrophoresis analysis of N protein purified through Ni-NTA columns. Lane 1, crude bacterial lysate; lanes 2 to 4, washes with 1, 2.5, and 5 mM imidazole, respectively; lane 5, purified recombinant N protein eluted by using 100 mM imidazole (indicated by arrow). Molecular masses (in kilodaltons) are indicated at the left.
FIG. 2
FIG. 2
Western blot analysis of induced bacterial cell lysates from different clones. Lanes 1 and 3, induced positive clones (clones 752 and 753, respectively) that express the recombinant ECV N protein; lanes 2 and 4, uninduced bacterial cell lysate controls; lane 5, negative control (induced bacterial lysate without the nucleoprotein gene). The arrow indicates the recombinant 50-kDa nucleocapsid protein.
FIG. 3
FIG. 3
Western blotting analysis of monoclonal antibodies to ECV N protein. Purified N protein was separated on a 10% polyacrylamide gel and electroblotted onto a nitrocellulose membrane. The strips were incubated with different anti-N protein monoclonal antibodies. Lanes 1 and 2, anti-histidine antibody and mouse anti-ECV polyclonal serum, respectively; lanes 3 to 7, different monoclonal antibody clones (clones 3C9, 7A4, 3C10, 8F2, and 9B8, respectively). The arrow indicates a 50-kDa N-protein band. The higher bands may be aggregates of ECV nucleoprotein expressed in E. coli.
FIG. 4
FIG. 4
Immunoprecipitation of ECV-infected cytoplasmic lysates. Human rectal tumor-18 cells were infected with ECV, and infected cytoplasmic extracts were immunoprecipitated with different anti-N protein monoclonal antibodies. The lysates were immunoprecipitated with monoclonal antibodies 7A4 (lane 1), 9B8 (lane 2), and 8F2 (lane 3). Lane 4, polyclonal serum against ECV used as a positive control; lane 5, mock-infected lysate used as a negative control; lane 6, protein molecular mass standard (in kilodaltons).
FIG. 5
FIG. 5
Immunohistochemical detection of BCV-antigen in formalin-fixed, paraffin-embedded tissue sections of bovine rectum. Plates a and b, plates c and d, and plates e and f are serial sections of the same region. Plates a, c, and e are immunostained with monoclonal antibody Z3A5, which recognizes the spike protein of BCV (16). Plates b, d, and f are immunostained with monoclonal antibody 8F2, which recognizes the nucleoprotein. Positive staining (red-brown stain) is present in the cytoplasms of infected crypt epithelial cells. The chromogen was 3,3′-diaminobenzidine; hematoxylin counterstain was used. Magnifications: ×328 (a to d) and ×374 (e and f).
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