Virology · 2025
Performance evaluation of micro-foci reduction neutralization test for the detection of neutralizing antibodies in human samples against dengue viruses circulating in India.
СтатьяОценочное исследованиеПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Chelluboina Shweta, Borkar Sneha, Chandra Mishra Akhilesh, Arankalle Vidya A, Shrivastava Shubham
Аннотация
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.
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.