Production and Characterization of Monoclonal Antibodies Against the Nucleocapsid Protein of SARS-COV

DNA encoding the N protein was amplified from cDNA prepared from the Urbani isolate (CDC) and cloned into the pBAD-TOPO protein expression vector (Invitrogen). The N protein was expressed in E. coli as a 6-His tagged fusion protein and purified using Ni-NTA columns (Qiagen). BALB/c mice were immunized with purified recombinant N protein at 2-week intervals for 8 weeks. Splenocytes from immunized mice were fused with NS-1 myeloma cells and cultured on 24-well plates. Cell culture supernatants from wells containing hybridoma colonies were initially screened by ELISA using recombinant N protein. Hybridoma cells from positive wells were subcloned, expanded, and retested. For Western blot analysis, Vero cells (ATCC) were mock-infected or infected with SARS-CoV (Urbani strain). The cell lysate was separated on a 15% polyacrylamide gel, transferred to PVDF membranes, and then incubated with an anti-N MAb. After extensive washing, the membranes were incubated with a HRP conjugated goat antimouse IgG (Jackson Laboratories). After 60 minutes, the blot was washed extensively

• Publication year

  2006

• Venue

  Advances in Experimental Medicine and Biology

• Publication date

  Unknown publication date

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1007/978-0-387-33012-9_27PMID 17037523PMCID 7139450
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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