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A custom-designed recombinant multiepitope protein as a dengue diagnostic reagent.
Protein Expr Purif. 2005 May; 41(1):136-47.PE

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+Show Affiliations

International Centre for Genetic Engineering and Biotechnology, New Delhi-110067, India.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15802231

Citation

AnandaRao, Ravulapalli, et al. "A Custom-designed Recombinant Multiepitope Protein as a Dengue Diagnostic Reagent." Protein Expression and Purification, vol. 41, no. 1, 2005, pp. 136-47.
AnandaRao R, Swaminathan S, Fernando S, et al. A custom-designed recombinant multiepitope protein as a dengue diagnostic reagent. Protein Expr Purif. 2005;41(1):136-47.
AnandaRao, R., Swaminathan, S., Fernando, S., Jana, A. M., & Khanna, N. (2005). A custom-designed recombinant multiepitope protein as a dengue diagnostic reagent. Protein Expression and Purification, 41(1), 136-47.
AnandaRao R, et al. A Custom-designed Recombinant Multiepitope Protein as a Dengue Diagnostic Reagent. Protein Expr Purif. 2005;41(1):136-47. PubMed PMID: 15802231.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A custom-designed recombinant multiepitope protein as a dengue diagnostic reagent. AU - AnandaRao,Ravulapalli, AU - Swaminathan,Sathyamangalam, AU - Fernando,Sirimali, AU - Jana,Asha M, AU - Khanna,Navin, PY - 2004/11/17/received PY - 2005/01/10/revised PY - 2005/4/2/pubmed PY - 2005/8/23/medline PY - 2005/4/2/entrez SP - 136 EP - 47 JF - Protein expression and purification JO - Protein Expr Purif VL - 41 IS - 1 N2 - 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. SN - 1046-5928 UR - https://www.unboundmedicine.com/medline/citation/15802231/A_custom_designed_recombinant_multiepitope_protein_as_a_dengue_diagnostic_reagent_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1046-5928(05)00011-2 DB - PRIME DP - Unbound Medicine ER -