Polyclonal antibodies to the bacterially expressed coat protein of Faba bean necrotic yellows virus

Specific rabbit polyclonal antibodies against bacterially expressed coat pr otein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were pro duced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE-9 (Qiagen, QI AGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N-termi nal 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. Ab out 8 mg of bacterially expressed CP (BE-CP) was purified from I litre of b acterial liquid culture using a Ni-NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl s ulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) was identified by i ts strong reaction with polyclonal antibodies produced against FBNYV partic les 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 intram uscular injections at weekly intervals. The antiserum produced was evaluate d for FBNYV detection in double antibody sandwich (DAS)-enzyme-linked immun osorbent assay (ELISA), triple antibody sandwich (TAS)-ELISA, tissue blot i mmunoassay 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 b ackground reactions with non-infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE-CP polyclonal antibody reacted weakl y 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 antibo dies were used. In addition, BE-CP polyclonal antibody reacted strongly wit h BE-CP in TAS-ELISA only when 2-5H9 detecting monoclonal was used. When mo noclonals were used as primary antibody and BE-CP polyclonal as detecting a ntibody (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 tissu e by TBIA and dot blot tests.