Production of monoclonal antibody against recombinant NS3 protein of bovine viral diarrhea virus (NADL strain)

Bovine Viral Diarrhea virus (BVDV) is an important viral pathogen of cattle causing several clinical syndromes. There are usually no pathognomonic clinical signs of BVDV infection. Diagnostic investigations therefore rely on serological detection and virus isolation. Nonstructural protein 3 (NS3) as immunogenic protein of BVDV is genetically and antigenically conserved among different isolates. This protein is therefore a candidate antigen for developing ELISA for serological studies. The aim of this study was to produce monoclonal antibody (MAb) against recombinant NS3 protein. For this purpose, the recombinant MBP-NS3 protein was expressed into expression vector pMalc2x in E. coli and purified using amylose resin chromatography column and the purified protein used as antigen in MAb production. After immunizing Balb/c mice with the recombinant antigen, the mouse showing highest titer of anti-NS3 antibody by indirect ELISA was selected for fusion. Spleen cells of the immunized mouse were fused with SP2/0 myeloma cells using polyethylene glycol. The cells in fusion mix were re-suspended in HAT medium and distributed in 96-well plates. Then culture supernatants of primary clones were screened by indirect ELISA. The positive clones after three times cloning, were selected and the reactivity of the monoclonal antibodies with recombinant and natural antigens was established by Western blotting. Based on these results, it appears that the specific monoclonal antibodies produced against NS3 recombinant antigen may be suitable for developing BVDV laboratory diagnostic assays.