Development of a novel NS1 Competitive Enzyme-Linked Immunosorbent Assay for the early detection of Zika virus infection

CASTILLO, DANIELA S.; ROLDÁN, JULIETA S.; CASSOLA, ALEJANDRO

Congreso; LXV Reunión anual de la Sociedad Argentina de Investigación Clínica; 2020

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 infection nor commercially available 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 is urgently needed for the detection of ZIKV acute infection. Recent studies have demonstrated that heat dissociation of the NS1 homo-hexamer, a useful diagnostic marker for flavivirus early detection, is a convenient alternative to enhance the sensibility of commercially available ELISAs by an increase in the antigen available monomeric forms. With this strategy in mind, we aimed to obtain monoclonal antibodies (mAbs) against denatured monomeric ZIKV NS1 (ZNS1) protein 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 two specifi¬c hybridoma clones, 1F5 and 6E2, which recognized the heat-denatured ZNS1 form by indirect ELISA. In addition, cross-reaction studies indicated that these mAbs specifically recognize a ZNS1 linear epitope, and that they do not cross-react with the NS1 protein from other related flaviviruses. The1F5 mAb enabled the development of a sensitive, reliable and reproducible icELISA to detect and quantify ZNS1 disease marker in heat-denatured human sera. We established a valid 1F5 based-icELISA that allows the detection and quantification of small amounts of ZNS1 (156 ng/ml), and constitutes a promising bioanalytical method for control strategies and the prevention of ZIKV propagation.