DEVELOPMENT OF HIGHLY SPECIFIC CAPTURE IMMUNOASSAYS FOR THE EARLY DETECTION OF ZIKA VIRUS INFECTION

ROLDÁN JS; CASSOLA A; CASTILLO DS

Mendoza

Congreso; LVIII Reunión anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular; 2022

Zika virus (ZIKV) is a flavivirus that is primarily transmitted from bites of infected Aedes aegypti or Aedes albopictus mosquitoes. There are currently no vaccines to prevent ZIKV infection nor commercially available clinical diagnostic tests demonstrated to identify ZIKV without cross-reactive interference of other related flaviviruses. In this context, the development of sensitive and accurate diagnostic methods is urgently needed for the early detection of ZIKV. Nonstructural protein 1 (NS1) is a highly conserved glycoprotein that is secreted as a hexamer and circulates at high levels in the bloodstream of acute patients. These properties turn ZIKV NS1 (ZNS1) into a good diagnostic marker, allowing early detection and diagnosis of ZIKV infection. In order to develop a highly specific and sensitive capture ELISA, we aimed at obtaining monoclonal antibodies (mAbs) against hexameric ZNS1 protein. We selected 6F6 specific hybridoma clone, which binds to a ZNS1 linear epitope. Cross-reaction studies through Western blotting, indirect ELISA and immunofluoresence staining indicated that 6F6 specifically recognizes ZNS1, and does not crossreact with the NS1 protein from other related flaviviruses. The 6F6 mAb enabled the development of a sensitive, reliable and reproducible capture ELISA with a limit of detection (LOD) of 10.8 ng/ml and a limit of quantification (LOQ) of 29.4 ng/ml. The accuracy of the 6F6 sandwich ELISA was assessed by spike-and-recovery tests, obtaining average recoveries between the ideal range from 80 to 120%. A quantitative capture ELISA was conducted using concentrated and inactivated supernatants of A549 cells infected with molecularly defined ZIKV or DENV1-4 isolates. We only detected 625 ng/ml of ZNS1 protein in the supernatant of A549 ZIKV infected cells, with no cross-reaction with DENV NS1 proteins. A customized lateral flow assay (LFA) kit was used to evaluate the suitability of the 6F6 mAb to perform as the capture and detection antibody in a sandwich LFA strip. The applied sample was normal human sera spiked with different concentrations of ZNS1. The minimum ZNS1 concentration which could be detected by the 6F6 LFA strip was 250 ng/ml. In conclusion, we established valid capture immunoassays that allow the detection and quantification of small amounts of ZNS1 in human sera, which constitute promising bioanalytical methods for control strategies and the prevention of ZIKV propagation.