Recombinant Multiepitope Protein for Early Detection of Dengue Infections

AnandaRao, Ravulapalli; Swaminathan, Sathyamangalam; Fernando, S.S.N.; Jana, Asha Mukul; Khanna, Navin

doi:10.1128/cvi.13.1.59-67.2006

Cited by 37 publications

(17 citation statements)

References 23 publications

(37 reference statements)

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“…Another assay for dengue diagnosis using recombinant proteins consisting of the N-terminal 80% of the envelopes of the four serotypes yielded 90% sensitivity and 86% specificity (6). Recently, results obtained using a protein composed of DENV E, NS1, and NS3 epitopes expressed in E. coli in ELISAs were consistent with those obtained with a commercial kit (1).…”

mentioning

confidence: 57%

Recombinant Polypeptide Antigen-Based Immunoglobulin G Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Dengue

Santos

Nogueira

Lima

et al. 2007

Clin Vaccine Immunol

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We have developed an indirect enzyme-linked immunosorbent assay for detection of anti-dengue virus (DENV) immunoglobulin G antibodies using four recombinant DENV envelope polypeptides as antigens, which demonstrated a sensitivity of 89.4% and a specificity of 93.3%. These easily produced antigens are a feasible, cost-effective alternative for generating reagents for dengue serological tests.The accurate and efficient diagnosis of dengue is important for clinical care, surveillance, pathogenesis studies, and vaccine research. Dengue viruses consist of four serotypes (DENV1 to -4) (17) and cause a spectrum of disease ranging from self-limited illness (dengue fever) to more severe forms of disease (dengue hemorrhagic fever/dengue shock syndrome) (25). Dengue is one of the most important mosquito-borne viral diseases in the world (10), with 2.5 to 3 billion people at risk for infection and tens of millions of dengue cases annually. Nonetheless, the disease remains significantly underreported (15). Although numerous commercial kits for serological diagnosis of dengue are available, some of which have shown good sensitivity and specificity (3,8), their cost poses a financial burden to many countries where dengue is endemic.Enzyme-linked immunosorbent assay (ELISA)-formatted tests to detect anti-DENV immunoglobulin G (IgG) antibodies have been developed as an alternative method to the hemagglutination inhibition test, which has served as the "gold standard" for the diagnosis and classification of DENV infections (4,5,12,18,25). Moreover, an IgG-ELISA was reported to be an accurate and reliable assay for characterization of human immune responses in primary and secondary DENV infections (18). However, these ELISAs require viral antigen produced, in limited quantities, by growing DENV in cell culture or in suckling mouse brain, which is insufficient for the large-scale serological screening made necessary by the spread of the disease. Furthermore, the use of whole viruses or crude extracts represents potential health hazards through exposure to infectious virus particles. To overcome this problem, we developed and evaluated an in-house ELISA for detecting anti-DENV IgG antibodies using as the antigen a mixture of DENV1 to -4 recombinant polypeptides, as its use for IgM capture has been previously demonstrated (23). Fragments of approximately 500 bp from the 5Ј end (nucleotides 1093 through 1585) of the DENV1 to -4 E genes were amplified by reverse transcription-PCR (RT-PCR) and cloned into an expression vector, and the immunogenic E polypeptides (ϳ25 kDa) were easily expressed in Escherichia coli (7, 23).The 271 sera used in this study were from the Flavivirus Laboratory, Oswaldo Cruz Institute/FIOCRUZ, Brazil (1998Brazil ( to 2005. Laboratory-positive DENV infection was defined in patients experiencing a febrile illness consistent with dengue according to WHO criteria (25), in accordance with which infection was confirmed by DENV isolation (9), detection of DENV RNA by RT-PCR (16), detection of anti-DENV IgM antibodie...

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confidence: 57%

Recombinant Polypeptide Antigen-Based Immunoglobulin G Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Dengue

Santos

Nogueira

Lima

et al. 2007

Clin Vaccine Immunol

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“…To characterize the purified rEDIII-T protein, it was subjected to SDS-PAGE, together with appropriate controls and prestained protein markers, electroblotted onto nitrocellulose, and probed with penta-His monoclonal antibody (MAb) and visualized using anti-mouse IgG-AP conjugate and BCIP/NBT substrate as described previously (1,2). To assess the reactivity of rEDIII-T protein towards anti-DEN virus IgM and IgG antibodies, the purified protein was electrophoresed in a single wide well and blotted onto nitrocellulose, which was then cut into narrow strips.…”

Section: Methodsmentioning

confidence: 99%

“…To this end, we performed a Western analysis in which the rEDIII-T protein, transferred onto nitrocellulose strips, was probed separately with several different antisera known to either contain or lack anti-DEN virus antibodies. We used sera that were characterized in our earlier studies (1,2). In this experiment, the binding of anti-DEN virus IgM and anti-DEN virus IgG antbodies in the sera to rEDIII-T was identified using anti-human IgM and anti-human IgG secondary anti- four DEN virus serotypes, and a sample lacking flaviviral antibodies.…”

Section: Design Of the Ediii-t Antigenmentioning

confidence: 99%

Single Antigen Detects both Immunoglobulin M (IgM) and IgG Antibodies Elicited by All Four Dengue Virus Serotypes

Hapugoda

Batra

Abeyewickreme

et al. 2007

Clin Vaccine Immunol

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The resurgence of dengue (DEN) virus infections in the last few decades coupled with the lack of a preventive vaccine and specific antiviral drugs has jointly contributed to making this a significant global public health problem. Currently, symptomatic supportive treatment and fluid replacement therapy are the only means available to minimize DEN-induced mortality. As the clinical symptoms associated with DEN virus infections are indistinguishable from those of many other viral, bacterial, and parasitic infections, specific diagnostic tests assume critical importance in the unequivocal identification of DEN virus infections. We have designed a novel chimeric antigen based on envelope domain III (EDIII), a critical antigenic region of the major structural protein of DEN viruses. We fused EDIIIs corresponding to each of the four DEN virus serotypes using pentaglycyl linkers, overexpressed the resultant tetravalent chimeric protein in Escherichia coli, and affinity purified it in high yields, obtaining ϳ30 mg protein of >95% purity per liter of culture. We show that this tetravalent antigen could specifically recognize anti-DEN virus antibodies of both the immunoglobulin M (IgM) and IgG classes. Using a large panel of IgM antibody capture-enzyme-linked immunosorbent assay-and hemagglutination inhibition-confirmed DEN virus-infected and uninfected patient sera (n ‫؍‬ 289), we demonstrate that this tetravalent antigen can function as a diagnostic tool of high sensitivity and specificity.Dengue (DEN) fever (DF), for which there is neither a vaccine nor any therapeutic drug, is currently the most important arboviral disease that places approximately half the global population at risk (16,35). While in the majority of cases, DF is clinically inapparent, it can progress in some cases to a severe hemorrhagic disease, dengue hemorrhagic fever (DHF), culminating in potentially fatal dengue shock syndrome (DSS) (11,14,17,35,43). DF and DHF/DSS are caused by infection with the mosquito-borne DEN viruses, of which there exist four antigenically distinct serotypes (DEN-1, -2, -3, and -4). These are members of the family Flaviviridae, which includes other flaviviruses such as yellow fever (YF), Japanese encephalitis (JE) and West Nile (WN) viruses (32). It is estimated that annually there are about 50 to 100 million cases of DF, of which ϳ500,000 progress to DHF, leading to ϳ25,000 deaths (12,(15)(16)(17). In untreated cases, mortality can be as high as 40 to 50% (20, 35). This can be significantly minimized by supportive care and symptomatic treatment through fluid replacement therapy (12). However, the combination of effective patient care and management rests on early definitive diagnosis of DEN virus infections. The clinical presentation of DF is often indistinguishable from those of other infectious diseases such as measles, influenza, typhoid, and other viral hemorrhagic fevers (11). In recent years, models have been developed to distinguish DF from other infections, based on clinical features and laboratory parameters suc...

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“…[3][4][5][6] The IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) is the assay of preference for the serological diagnosis of acute dengue virus infection, and is a very useful tool for sero-epidemiological dengue surveillance. 7,8 The detection of both anti-dengue IgM and IgG by ELISA permits classification of the dengue infection type (primary or secondary), thereby contributing to a better understanding of the pathogenesis of DHF.…”

Section: Introductionmentioning

confidence: 99%

Monoclonal antibody to dengue capsid protein

Vazquez¹,

Pupo-Antúnez²,

Vázquez³

et al. 2009

mAbs

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Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection.

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Recombinant Multiepitope Protein for Early Detection of Dengue Infections

Cited by 37 publications

References 23 publications

Recombinant Polypeptide Antigen-Based Immunoglobulin G Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Dengue

Recombinant Polypeptide Antigen-Based Immunoglobulin G Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Dengue

Single Antigen Detects both Immunoglobulin M (IgM) and IgG Antibodies Elicited by All Four Dengue Virus Serotypes

Monoclonal antibody to dengue capsid protein

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