Comparison of Plaque- and Enzyme-Linked Immunospot-Based Assays To Measure the Neutralizing Activities of Monoclonal Antibodies Specific to Domain III of Dengue Virus Envelope Protein

Abstract

The plaque reduction neutralization test (PRNT) is used widely to measure the neutralization activity of anti-dengue virus (DENV) antibodies, but it is time-consuming and labor-intensive and has low sample throughput. For fast and convenient measurement of neutralizing antibodies, especially in evaluating the efficiency of the DENV vaccines on a large scale, a new method is needed to replace PRNT. In recent decades, several microneutralization assays have been developed to overcome the limitations of PRNT. In the present study, we evaluated one of these, the enzyme-linked immunospot microneutralization test (ELISPOT-MNT), in comparison with PRNT. ELISPOT-MNT is performed in 96-well format, and the plaques are developed after 2 to 4 days using an ELISA to transform them into spots, which are detected automatically with an ELISPOT instrument. The assay is faster than PRNT, has a high throughput, and is more objective. We used 10 monoclonal antibodies (MAbs) against domain III of the DENV envelope protein (EDIII) to evaluate the two assays; all of these MAbs cross-react with all four serotypes of DENV as measured by immunofluorescence assay. The two neutralization assays were performed simultaneously to measure the 50% inhibitory concentration (IC 50 ) of these MAbs. Using PRNT as the reference and treating IC 50 values higher than 50 g/ml of MAbs as negative, ELISPOT-MNT showed a sensitivity of 95.6% and specificity of 88.24% when 10 MAbs were tested against four DENV serotype strains. A good correlation (R 2 ‫؍‬ 0.672; P ‫؍‬ 0.000) was observed between the two assays, making ELISPOT-MNT a potentially valuable method for measure of neutralizing antibodies against DENV. D engue virus (DENV) is a mosquito-borne virus that belongs to the Flavivirus genus in the Flaviviridae family (11). DENV has four known serotypes: DENV-1, DENV-2, DENV-3, and DENV-4. Infection with any of the four serotypes can cause a spectrum of diseases ranging from dengue fever (DF) to dengue hemorrhagic fever/ dengue shock syndrome (DHF/DSS) (4). In the absence of effective vaccines or specific treatments, dengue has become a major public health problem throughout the tropical and subtropical areas of the world (18). Antibodies elicited by one primary DENV serotype infection are not strongly protective against the other three; conversely they may lead to the development of DHF or DSS because the crossreactivity may facilitate viral infection through Fc receptor-mediated binding to monocytes (5, 6). For this reason, any dengue vaccine produced must be evaluated for its ability to induce long-term and simultaneous protection against all four serotype DENV, in order to avoid antibody-dependent enhancement (ADE) of viral infection. Therefore, in vaccine research, the protective ability of each antibody needs to be evaluated.The plaque reduction neutralization test (PRNT) has been considered the gold standard for detecting the neutralization activity of antibodies against DENV since it was first introduced in 1967 (14). Although WHO has developed a...

Evaluation of Commercially Available Assays for Diagnosis of Acute Dengue in Schoolchildren During an Epidemic Period in Medellin, Colombia

“…8,21,23,26 A recent study showed that ELISPOT-MNT had a sensitivity of 95.6% and specificity of 88.2%, with a good correlation (R 2 = 0.7) with PRNT. 16 Because of its very high sensitivity, the use of ELISPOT-MNT as the comparative standard method in our study leads inevitably to lower statistics performances of the assays that are compared with it, which can explain the relatively low performances of both diagnostic strategies.…”

“…14 However, standard methods such as HI and PRNT are labor intensive, time consuming and require acute and convalescent sera with an interval of more than 7 days, which makes them inadequate for routine early diagnosis. 15 Thus, 16 In 2010, Colombia experienced the worst dengue outbreak in its history. During this epidemic year and the following post-epidemic period, an active surveillance study of dengue cases in schoolchildren was performed in the Colombian City of Medellin.…”

Diagnosis of dengue: an update

“…These new tests have been separately validated, compared and evaluated [124,[126][127][128][129][130] against PRNT and found to have good agreement (false-positive rate <10%) in cases with primary DENV infection [124,130]. However, Putnak et al reported poor agreement among the tests in association with vaccination or secondary infection [124].…”

Risk Factors Associated with Dengue Transmission and Spatial Distribution of High Seroprevalence in Schoolchildren from the Urban Area of Medellin, Colombia

“…Enzyme-linked immunospot and microneutralization tests (ELISPOT-MNT) [21] were performed in 278 serum samples following a standardized previously published protocol [22]. The ELISPOT-MNT 50 titer was determined as the serum dilution that reduces the number of foci or plaques by at least 50% when compared with wells with virus control.…”