Development of a novel NS1 competitive enzyme-linked immunosorbent assay for the early detection of Zika virus infection

Zika virus (ZIKV) is a flavivirus that has emerged as a global health threat after the 2015 outbreak in the Americas, where devastating congenital defects were documented. There are currently no vaccines to prevent ZIKV infections nor commercially available clinical diagnostic tests demonstrated to identify ZIKV without cross-reactive interference of related flaviviruses. Early diagnosis is critical when treating symptomatic patients and in preventing ZIKV transmission. In this context, the development of sensitive and accurate diagnostic methods are urgently needed for the detection of ZIKV acute infection. The aim of this study consisted of obtaining monoclonal antibodies (mAbs) against denatured monomeric ZIKV Nonstructural protein 1 (ZNS1), a useful diagnostic marker for flavivirus early detection, in order to develop a highly specific and sensitive ZNS1 indirect competitive ELISA (icELISA). The production of hybridomas secreting ZNS1 mAbs was carried out through immunizations with denatured monomeric ZNS1. We selected 1F5 and 6E2 hybridoma clones, which recognized the heat-denatured ZNS1 hexameric form by indirect ELISA. Cross-reaction studies indicated that these mAbs specifically bind to a ZNS1 linear epitope, and that they do not cross-react with the NS1 protein from other related flaviviruses. The 1F5 mAb enabled the development of a sensitive and reproducible icELISA to detect and quantify small amounts of ZNS1 disease marker in heat-denatured human sera. Here, we establish a reliable 1F5 based-icELISA that constitutes a promising diagnostic tool for control strategies and the prevention of ZIKV propagation.