Microneutralization assay for detection of antibodies against dengue virus serotype 2

Keywords: Dengue, Serotype, Neutralization, Optimization, Immunoglobulin


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.


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Sevilla Drozdek LA, Mayta Huatuco E, Mamani Zapana E, Reyes Puma N, Vásquez Cajachahua K, Quispe Bravo B, Hermosilla Jara J, Quintana Bedoya A, Sulca Herencia J. Microneutralization assay for detection of antibodies against dengue virus serotype 2. Acta Med Peru [Internet]. 2023Aug.2 [cited 2024May25];40(2). Available from: https://amp.cmp.org.pe/index.php/AMP/article/view/2538