Microneutralization assay for detection of antibodies against dengue virus serotype 2

Abstract

Objective: Adapt the microneutralization test (MNT) to detect neutralizing antibodies against dengue serotype 2 virus (DENV-2) in the Vero-76 cell line.  Materials and methods. Different cellular concentrations (0.6 x 10⁵ cell / mL, 0.9 x 10⁵ cell / mL, 1.2 x 10⁵ cell / mL) and percentages of fetal bovine serum (FBS) at 2%, 3% and 4%, passages number of virus stock and incubation days were evaluated. The viral seed was confirmed by RT-qPCR. DENV-2 was propagated by carrying out 5 successive passages in the Vero-76 cell line, then the virus was titrated in microplates and 2 methods were evaluated: complete monolayer and cell suspension, and the optimal coloring day was determined.  Obtained the results, it was evaluated by the MNT for DENV-2: 5 sera negative to DENV-2 and YFV, 5 sera negative to DENV-2 and seropositive to YFV and 5 seropositive sera to DENV-2 confirmed by plaque reduction neutralization test.  The samples were evaluated in triplicate and virus control and cell control were used. Results: The optimal method for MNT uses cells in suspension (0.9 x 10⁵ cell / mL), 2% SBF and viral seed passage 5. The minimum dilution capable of differentiating a DENV-2 seropositive serum from a DENV-2 seronegative sera was 1:40, and the incubation time of the MNT for DENV-2 was 10 days. Conclusion: MNT allows the detection of IgG antibodies against DENV-2, provides reliable results and a greater number of samples can be analyzed with material savings.