Although it is considered the reference for quantification of neutralizing antibodies, classical method of the plaque reduction neutralization test (PRNT) is labor intensive, requires specific equipment and inputs, besides a long time for its finalization, even in the micro-PRNT version (in 96-well plates). It has a higher sample throughput, however the smaller wells make the reading of plaques more difficult. With an immunoenzymatic revelation step and a semi-automated reading, the μFRN-HRP (micro Focus Reduction Neutralization - Horseradish Peroxidase) is a faster and more efficient test for the quantification of YF neutralizing antibodies. This study aimed to standardize, validate, and compare it with the reference method in 6-well plates (PRNT). Once the execution protocol was standardized, precision, accuracy, selectivity, and robustness were evaluated to validate the μFRN-HRP. In addition, 200 sera of vaccinees were processed by the μFRN-HRP and by the micro-PRNT to compare with the reference test, estimating agreement by Intraclass Correlation Coefficient (ICC). The standardization and validation of the μFRN-HRP was carried out successfully. Weak to moderate agreement was observed between μFRN-HRP and PRNT for titers in reciprocal dilution, while the same comparison between the classical tests resulted in a better ICC. However, titers in milli-international units obtained by μFRN-HRP showed a substantial agreement with PRNT, while the agreement between micro-PRNT and PRNT was inferior. Therefore, μFRN-HRP can be used in the confirmation of natural YF infection and immune response to vaccination, replacing the micro-PRNT, gaining agility, while preserving the specificity of the result.
Keywords: FRNT; Neutralizing antibodies; PRNT; Standardization; Validation; Yellow fever virus.
Copyright © 2023. Published by Elsevier B.V.