Plaque reduction neutralization test (PRNT₅₀) for the detection of anti-yellow fever antibodies from clinical samples

Yellow fever (YF) is a mosquito-borne viral haemorrhagic disease that remains endemic in parts of South America and Africa. Although there is a safe and effective vaccine that has been available for over 75 years, YF remains a public health problem in areas with endemic as well as sporadic transmission. Immune responses elicited by natural infection with yellow fever virus and vaccination are marked by production of neutralizing antibodies. Serological cross-reactivity exhibited by flaviviruses poses challenges to the diagnostic tests for detection of YF-specific antibodies. The most specific plaque reduction neutralization test (PRNT) is considered the "gold standard" test for detecting and measuring the neutralizing antibodies. This study was undertaken to develop and validate in-house PRNT₅₀ test for measurement of YF specific neutralizing antibodies. We validated PRNT₅₀ test using different parameters including specificity, linearity, precision, accuracy and robustness. The YF PRNT₅₀ assay was shown to be specific, robust, precise and accurate. Thus, we have proven suitability of this assay for evaluation of neutralizing antibodies in response to YF vaccination in case of natural infections.