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

Abstract

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...

Polyclonal antibodies against properly folded Dengue virus NS1 protein expressed in E. coli enable sensitive and early dengue diagnosis

“…Because rapid and accurate diagnosis of dengue is critical for appropriate treatment, different approaches have been developed to generate a sensitive acute-phase diagnostic assay (dos Santos et al, 2004Santos et al, , 2007. It is known that the NS1 protein is secreted into the patient's plasma at the beginning of clinical symptoms, mostly from days 1 to 6 after the onset of fever (Alcon et al, 2002), which makes NS1 an important marker for detecting DENV infection in the first few days after symptoms appear.…”

A Chikungunya Virus Multiepitope Recombinant Protein Expressed from the Binary System Insect Cell/Recombinant Baculovirus Is Useful for Laboratorial Diagnosis of Chikungunya

“…The MULTREC IgG-ELISA was performed, as previously described by Dos Santos et al [ 44 ], with modifications. Microplates of 96-wells (Immulon Dynatech Industries, Inc., Chantilly, VA, USA) were coated with 100 µL of a mixture of recombinant CHIKV multi-epitope gene (R1) (6 µg/mL each per well) and incubated overnight at 4 °C.…”

“…The MULTREC IgG-ELISA was performed, as previously described by Dos Santos et al [44], with modifications. Microplates of 96-wells (Immulon Dynatech Industries, Inc., Chantilly, VA, USA) were coated with 100 µL of a mixture of recombinant CHIKV multiepitope gene (R1) (6 µg/mL each per well) and incubated overnight at 4 • C. The plates were blocked with 1× PBS pH 7.4, 5% nonfat drink milk, 3% goat serum, 3% fetal bovine serum (FBS), 1% BSA, and 20% Tween 20 for 1 h at 37 • C. One hundred microliters of 1:40 diluted patients' serum was added with the same blocking solution; the plates were incubated at 37 • C for 2 h. Posteriorly, 40 µL of conjugated anti-human IgG with horseradish peroxidase (Sigma-Aldrich) diluted 1:5000 was added.…”

Dengue Fever in Rio: Macumba versus Voltaire

“…That gallery came to life a century ago when Brazil founded its Instituto Oswaldo Cruz to put "science at the service of the Brazilian people." Its scientists busy themselves with the newest devices of molecular biology to map the dengue virus and make its diagnosis neat and rapid (27). Finally, as this is written, the federal government of Brazil has over-ridden the mayor and is moving its batallions of health workers into Rio to kill mosquitos and cleanse the favelas.…”