Multisite Monoclonal Immunoassay for Dengue Viruses: Detection of Viraemic Human Sera and Interference by Heterologous Antibody

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Abstract
A monoclonal radioimmunoassay (RIA) was developed for detection of dengue virus in infected cell culture fluids and blood samples from dengue patients. Antibodies used to construct the RIA were selected on the basis of high binding avidity, the demonstration of synergism in competitive binding assays and empirical trials with different antibody combinations. Optimal binding of all four dengue virus serotypes was achieved by use of a flavivirus group-reactive and a dengue virus complex-reactive antibody attached to a solid-phase support and a single flavivirus subgroup-reactive antibody as radiolabelled probe. A ''simultaneous sandwich'' format and prolonged (18 h) incubation at 37.degree. C yielded optimal results. The limit of sensitivity of the RIA for detection of dengue type 2 virus was 2.7 log10 mosquito 50% infectious doses (MID50). The assay was tenfold more sensitive for dengue type 2 than for dengue types 1 and 3 viruses and 100-fold more sensitive than for dengue type 4 virus. Specificity, assessed using over 500 disease control human sera, was increased by addition of monoclonal anti-tetanus blocking antibodies, resulting in a false positive rate of only 0.2%. Heterologous dengue virus antibodies were shown to inhibit the RIA in assays performed with artificial immune complexes. Acute phase human sera containing 104.2 to 107.6 MID50 but no detectable antigen by RIA, were also shown to inhibit binding of the homologous dengue virus serotype; this effect was attributed to heterologous antibody from a prior infection. Among 116 viraemic sera from dengue patients, the RIA was positive in 43 to 47% of patients with dengue type 1, 2 or 3 infections but in only 10% of the dengue type 4 cases. Virus was most frequently detected in cases of primary infection (54%) than in cases of superinfection (16%). Despite the limitations imposed by immunological interference, the antigen capture RIA appears useful as a rapid diagnostic technique for dengue surveillance.

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