Standardization, validation, and comparative evaluation of a faster and high-performance test for quantification of yellow fever neutralizing antibodies

Abstract

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.