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

Abstract

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.

Fig 1. Relative affinity of 1F5 and 6E2 mAbs for the denatured monomeric and the native hexameric ZNS1 conformations.

Standard curves of denatured monomeric or native hexameric HEK293-expressed ZNS1 detected by iELISA with (A) 1F5 or (B) 6E2 mAbs. Each point of the curve represents mean±SEM of three sample replicates. IC50 values of the mAbs are indicated.

Fig 2. Western blot analysis of flavivirus recombinant NS1 proteins with 1F5 and 6E2 mAbs.

SDS-PAGE analysis of E.coli-expressed DENV1-4, JEV, SLEV, TBEV, USUV, WNV, YFV and ZIKV recombinant NS1 protein samples analyzed by immunoblot using (A) 1F5 or (B) 6E2 mAbs, or by (C) Coomassie brilliant blue staining. The position of the molecular mass standards is indicated on the left.

Fig 3. Comparative reactivity of flavivirus recombinant NS1 proteins by indirect ELISA with 1F5 and 6E2 mAbs.

Standard curves of denatured E.coli-expressed DENV1-4, JEV, SLEV, TBEV, USUV, WNV, YFV and ZIKV recombinant NS1 proteins by iELISA with (A) 1F5 or (B) 6E2 mAbs. Each point of the curve represents mean±SEM of three sample replicates. IC50 and CR values of the mAbs are indicated. N.A.: not applicable.

Fig 4. Cross-reactivity analysis of 1F5 and 6E2 mAbs against different flaviviruses in infected cells.

Immunostaining of ZIKV and DENV1-4 A549 infected cells with (A) 1F5 or (B) 6E2 mAbs. Uninfected A549 cells (mock) were used as the negative control. Scale bar, 25 μm.

Fig 5. Standard curve for the detection of ZNS1 by indirect competitive ELISA with 1F5 mAb.

Standard curve of the icELISA to detect denatured ZNS1 using 1F5 mAb, calculated with a 4 parameter logistic regression fitting with the following equation: Y = Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*Hill slope)), where Bottom = 5.28, Top = 100.6, EC50 = 1.26 and Hill slope = 2.87. Each point of the curve represents mean±SEM of six sample replicates.