title: Production of IgM specific recombinant dengue multiepitope protein for early diagnosis of dengue infection.
abstract: Dengue virus infections have recently undergone dramatic expansion in range, affecting several tropical and subtropical regions of the world. Early detection of dengue infection based on the identification of antibodies has emerged as a practical and reliable means of diagnosis of dengue fever. The recombinant dengue multiepitope (rDME-M) protein specific to IgM in E. coli was produced in a 5-L fermentor for use in diagnostic purpose. After fermentation, dry cell weight was approximately 11.8 g/L of the culture. The rDME-M protein was purified under denaturing conditions using single-step nickel nitrilotriacetate (Ni-NTA) affinity chromatography. The final yield of purified rDME-M protein from this method was approximately 68.5 mg/L of the culture. The purity of rDME-M protein was checked by SDS-PAGE analysis, and the reactivity of this protein was further checked by Western blotting and enzyme-linked immunosorbent assay (ELISA). The purified protein was used as an antigen in the development of an in-house dipstick ELISA and evaluated with a panel of 80 patient sera, characterized using commercially available tests for detection of dengue antibody. The results were in excellent agreement with those of IgM capture ELISA (Pan-Bio) and rapid immunochromatography (IC) test (Pan-Bio). These results show that the in-house dipstick ELISA using rDME-M protein can be used as a promising kit because of its comparable sensitivity, specificity, field applicability, and low cost.
authors.affiliation: Division of Virology, Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, India. tripathink@gmail.com
authors.forename: Nagesh K
authors.initials: NK
authors.lastname: Tripathi
authors.forename: Ambuj
authors.initials: A
authors.lastname: Shrivastva
authors.forename: Priyabrata
authors.initials: P
authors.lastname: Pattnaik
authors.forename: Manmohan
authors.initials: M
authors.lastname: Parida
authors.forename: Paban K
authors.initials: PK
authors.lastname: Dash
authors.forename: Nimesh
authors.initials: N
authors.lastname: Gupta
authors.forename: Asha M
authors.initials: AM
authors.lastname: Jana
authors.forename: P V Lakshmana
authors.initials: PV
authors.lastname: Rao
mesh_terms: D003715:Dengue / Q000175:diagnosis* / Q000276:immunology; D003716:Dengue Virus / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007075:Immunoglobulin M / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology*; D014764:Viral Proteins / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000939:Epitopes; D007075:Immunoglobulin M; D011994:Recombinant Proteins; D014764:Viral Proteins