title: Production and characterization of monoclonal antibody specific to recombinant dengue multi-epitope protein.
abstract: Monoclonal antibodies against novel dengue recombinant protein were produced following immunization of Balb/c mice with recombinant dengue multi-epitope protein (r-DMEP) expressed in Escherichia coli vector and purified in a single-step chromatography system. Antigenicity of r-DMEP was evaluated by dot enzyme immunoassay. Mice were immunized intraperitoneally with five doses each of 100 microg of this novel antigen at 1-week intervals and a final intravenous booster dose prior to the fusion. Hybridomas resulted from fusion of myeloma cells and splenocytes using PEG-1500 as an additive. Selection of the hybrids was done using HAT medium, and the hybrids thus selected were finally screened qualitatively and quantitatively by dot and plate immunoassays, respectively. Five antibody secretory hybrid clones exhibited specific reactivity against r-DMEP by dot-ELISA, whereas a lone clone was found to be cross-reactive with Japanese encephalitis virus (JEV). Monoclonal antibodies (MAbs) specific to r-DME protein recognized the envelope and non-structural epitopes by Western blot analysis. These MAbs were further checked for their diagnostic efficacy using dengue suspected clinical samples and found overall sensitivity and specificity for DRDE dipstick ELISA. MAb-based dipstick ELISA results were 85%, 75% and 85%, 90%, respectively.
authors.affiliation: Division of Virology, Defence Research and Development Establishment, Gwalior, India. abhyankarjay@gmail.com
authors.forename: Ajay Vinayak
authors.initials: AV
authors.lastname: Abhyankar
authors.forename: Rakesh
authors.initials: R
authors.lastname: Bhargava
authors.forename: Asha Mukul
authors.initials: AM
authors.lastname: Jana
authors.forename: Ajay Kumar
authors.initials: AK
authors.lastname: Sahni
authors.forename: P V Lakshmana
authors.initials: PV
authors.lastname: Rao
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D003001:Cloning, Molecular; D006224:Cricetinae; D003716:Dengue Virus / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry*; D006825:Hybridomas / Q000378:metabolism; D007118:Immunoassay / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D009101:Multiple Myeloma / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013154:Spleen / Q000166:cytology; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D014764:Viral Proteins