Polyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virus uri icon

abstract

  • Specific rabbit polyclonal antibodies against bacterially expressed coat protein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were produced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE-9 (Qiagen, QIAGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N-terminal hexahistidine tag in Escherichia coli M 15 cells was induced by adding isopropyl-3-D-1-thiogalactoside (IPTG) to a final concentration of 2 mM. About 8 mg of bacterially expressed CP (BE-CP) was purified from I litre of bacterial liquid culture using a Ni-NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against FBNYV particles and 2-5H9 FBNYV-monoclonal in Western blots. Expressed and purified CP (SDS-PAGE 23 kDa band) was injected into a white rabbit, using seven intramuscular injections at weekly intervals. The antiserum produced was evaluated for FBNYV detection in double antibody sandwich (DAS)-enzyme-linked immunosorbent assay (ELISA), triple antibody sandwich (TAS)-ELISA, tissue blot immunoassay JBIA), dot blot, Western blot and goat antimouse coating (GAMC)-ELISA using 13 different FBNYV monoclonal antibodies. The antiserum raised against the BE-CP gave strong FBNYV-specific TBIA reactions and very weak background reactions with non-infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE-CP polyclonal antibody reacted weakly with FBNYV-infected tissue and strongly with BE-CP in DAS-ELISA, but not with FBNYV-infected tissue in TAS-ELISA when 13 detecting monoclonal antibodies were used. In addition, BE-CP polyclonal antibody reacted strongly with BE-CP in TAS-ELISA only when 2-5H9 detecting monoclonal was used. When monoclonals were used as primary antibody and BE-CP polyclonal as detecting antibody (GAMC-ELISA), FBNYV-infected tissue gave moderate reactions with 2-5H9 and strong reactions with 3-2E9 monoclonal, whereas BE-CP gave equally strong reactions with both monoclonals. These results showed that the BE-CP polyclonal antibody is useful for the detection of FBNYV in infected tissue by TBIA and dot blot tests.

publication date

  • 2001
  • 2001