Detection of Serotype-Specific Antibodies to the Four Dengue Viruses Using an Immune Complex Binding (ICB) ELISA

Abstract

Dengue virus (DENV) infections are preferentially diagnosed by detection of specific IgM antibodies, DENV NS1 antigen assays or by amplification of viral RNA in serum samples of the patients. The type-specific immunity to the four worldwide circulating DENV serotypes can be determined by neutralization assays. An alternative to the complicated neutralization assays would be helpful to study the serotype-specific immune response in people in DENV hyperendemic areas but also in subjects upon DENV vaccination. In consecutive samples of patients with DENV-1- 4 infection type-specific antibodies were detected using an immune complex binding (ICB) ELISA. During incubation of serum samples and enzyme- labeled recombinant envelope domain III (EDIII) antigens immune complexes (ICs) are formed, which are simultaneously bound to a solid phase coated with an Fc–receptor (CD32). After a single washing procedure the bound labeled ICs can be determined. To further improve type-specific reactions high concentrations of competing heterologous unlabeled ED III proteins were added to the labeled antigens. Follow-up serum samples of 64 patients with RT-PCR confirmed primary DENV-1, -2, -3 or -4 infections were tested against four enzyme-labeled recombinant DENV EDIII antigens. Antibodies to the EDIII antigens were found in 55 patients (sensitivity 86%). A complete agreement between the serotype detected by PCR in early samples and the serotype-specific antibody in later samples was found. Type-specific anti-EDIII antibodies were first detected 9–20 days after onset of the disease. In 21% of the samples collected from people in Vietnam secondary infections with antibodies to two serotypes could be identified. The data obtained with the ICB-ELISA show that after primary DENV infection the corresponding type-specific antibodies are detected in almost all samples collected at least two weeks after onset of the disease. The method will be of value to determine the distribution of the various type-specific anti–DENV antibodies in DENV endemic areas. Infections with four different dengue viruses are threatening 2.5 billion people in tropical countries. Since most antibodies to these four viruses are cross-reacting, a type-specific ELISA would be valuable to study the immune response to the circulating viruses in patients but also in healthy subjects in endemic counties. Therefore a novel DENV immune complex binding (ICB) ELISA was developed to detect serotype-specific antibodies to all four dengue virus serotypes in human serum samples. The tests use labeled recombinant EDIII antigens of the four DENV strains. Numerous samples of patients with RT-PCR confirmed dengue fever were assessed by the new method. In samples of 55 patients with primary dengue fever full agreement between the serotype detected by RT-PCR and the serotype-specific antibody based on the ICB ELISA was obtained. The type-specific antibodies were not observed before the second week of illness. Our data suggest that using the ICB ELISA in healthy adult subjects in an endemic region (Vietnam) both primary and secondary infections can be identified. The method may help to analyze the distribution of the four dengue viruses in the tropics.