title: A custom-designed recombinant multiepitope protein as a dengue diagnostic reagent.
abstract: Currently, dengue fever is the most important re-emerging mosquito-borne viral disease, with the major proportion of the target population residing in the developing countries of the world. In endemic areas, potentially fatal secondary dengue infections, characterized by high anti-dengue IgG antibody titers, are most common. Most currently available commercial dengue diagnostic kits rely on the use of whole virus antigens and are consequently associated with false positives due to serologic cross-reactivity, high cost of antigen production, and biohazard risk. This has prompted the need to develop an alternate antigen to replace the whole virus antigen in diagnostic tests. We have designed and expressed a novel recombinant protein antigen by assembling key immunodominant linear IgG-specific dengue virus epitopes, chosen on the basis of pepscan analysis, phage display, and computer predictions. The recombinant dengue multiepitope protein was expressed to high levels in Escherichia coli, purified in a single step, yielding >25 mg pure protein per liter culture. We developed an in-house enzyme-linked immunosorbent assay (ELISA) to detect anti-dengue antibodies in a panel of 20 patient sera using the purified recombinant dengue multiepitope protein as the capture antigen. The ELISA results were in excellent agreement with those obtained using a commercially available diagnostic test, Dengue Duo rapid strip test from PanBio, Australia. The high epitope density, careful choice of epitopes, and the use of E. coli system for expression, coupled to simple purification, jointly have the potential to lead to the development of an inexpensive diagnostic test with a high degree of sensitivity and specificity.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, New Delhi-110067, India.
authors.forename: Ravulapalli
authors.initials: R
authors.lastname: AnandaRao
authors.forename: Sathyamangalam
authors.initials: S
authors.lastname: Swaminathan
authors.forename: Sirimali
authors.initials: S
authors.lastname: Fernando
authors.forename: Asha M
authors.initials: AM
authors.lastname: Jana
authors.forename: Navin
authors.initials: N
authors.lastname: Khanna
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis; D000956:Antigens, Viral / Q000235:genetics; D003715:Dengue / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D003716:Dengue Virus / Q000145:classification / Q000235:genetics* / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005814:Genes, Viral; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D007202:Indicators and Reagents; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D012703:Serotyping; D014764:Viral Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D000939:Epitopes; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D014764:Viral Proteins