Evaluation of three alternative methods to the plaque reduction neutralizing assay for measuring neutralizing antibodies to dengue virus serotype 2

Abstract

Background: Dengue is a global public health challenge which requires accurate diagnostic methods for surveillance and control. The gold standard for detecting dengue neutralizing antibodies (nAbs) is the plaque reduction neutralization test (PRNT), which is both labor-intensive and time-consuming. This study aims to evaluate three alternative approaches, namely, the MTT-based (or (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) microneutralization assay, the xCELLigence real-time cell analysis (RTCA), and the immuno-plaque assay-focus reduction neutralization test (iPA-FRNT).

Methods: Twenty-two residual serum samples were tested for DENV-2 nAbs using all four assays at three neutralization endpoints of 50%, 70% and 90% inhibition in virus growth. For each neutralization endpoint, results were compared using linear regression and correlation analyses. Test performance characteristics were further obtained for iPA-FRNT using 38 additional serum samples.

Results: Positive correlation of DENV-2 neutralization titers for the MTT-based microneutralization assay and the PRNT assay was only observed at the neutralization endpoint of 50% (r = 0.690). In contrast, at all three neutralization end points, a linear trend and positive correlation of DENV-2 neutralization titers for the xCELLigence RTCA and the PRNT assays were observed, yielding strong or very strong correlation (r = 0.829 to 0.967). This was similarly observed for the iPA-FRNT assay (r = 0.821 to 0.916), which also offered the added advantage of measuring neutralizing titers to non-plaque forming viruses.

Conclusion: The xCELLigence RTCA and iPA-FRNT assays could serve as suitable alternatives to PRNT for dengue serological testing. The decision to adopt these methods may depend on the laboratory setting, and the utility of additional applications offered by these technologies.

Keywords: Dengue; Neutralizing antibodies; PRNT; Plaque reduction neutralization test; Serology.

Fig. 1

Neutralizing DENV antibody titers against DENV-2 Cosmopolitan genotype virus strain using the PRNT assay. DENV-2 neutralizing antibody (nAb) titers for 22 serum samples at three levels of virus neutralization endpoints (samples 1–19: Dengue IgG positive; samples 20–22: Dengue IgG negative)

Fig. 2

Comparison of DENV-2 neutralization titers obtained by the MTT-based microneutralization, the xCELLigence RTCA and the iPA-FRNT assays with the standard PRNT assay. Linear regression and scatterplot of neutralizing titers comparing the evaluated assay with standard PRNT at neutralization endpoints of 50% (left panel), 70% (middle panel) and 90% (right panel). Panel A-C: MTT-based microneutralization assay. Non-linearity was observed for two of three neutralization endpoints (NT₇₀ and NT₉₀). (D-F) xCELLigence RTCA assay. (G-I) iPA-FRNT assay. At all three neutralization endpoints, positive correlation was observed for both the xCELLigence RTCA and iPA-FRNT assays