Performance evaluation of micro-foci reduction neutralization test for the detection of neutralizing antibodies in human samples against dengue viruses circulating in India.

BACKGROUND

Dengue vaccine studies largely hinge on the understanding of the serostatus of a population by measuring the levels of neutralizing antibodies against all four dengue serotypes. In this study, we optimized the micro-foci-reduction neutralization test (micro-FRNT) that measures neutralizing antibody titres in a 96-well plate format.

METHODS

A total of 53 healthy blood donor serum/plasma samples, (31 anti-dengue IgG positive and 22 anti-dengue IgG negative) were used to validate the micro-FRNT against circulating Indian strains of all four serotypes of dengue viruses (DENV). Key parameters such as robustness, accuracy, precision, sensitivity, and specificity were examined. In a subset of 30 samples, the neutralization test was performed simultaneously in 24-well and 96-well plate formats, and FRNT50 titres were compared.

RESULTS

Our results demonstrated that 100%, 87%, 100% and 100% of samples had micro-FRNT50 titres within 25% CV in the intra-assay precision against DENV-1, -2, -3 and -4, respectively. The micro-FRNT demonstrated inter-assay precision within 30% CV in 100%, 80% of samples against DENV-1/-2/-3 and DENV-4 serotypes, respectively. The lower limit of quantitation for DENV-1, DENV-2, DENV-3, and DENV-4 was determined to be 16, 13, 11, and 11, respectively. We also demonstrated comparable FRNT50 titres using a 96-well plate against the standard 24-well plate format, yielding excellent positive correlation (r = 0.88 to 0.96) for all four serotypes.

CONCLUSION

In summary, we demonstrated the validation of the DENV-specific micro-FRNT method for DENV with a reduced turnaround time. The micro-FRNT method presents a useful tool for monitoring immune response against dengue viruses during epidemiological and vaccine response studies.