Development of a Rapid Diagnostic Test Kit to Detect IgG/IgM Antibody against Zika Virus Using Monoclonal Antibodies to the Envelope and Non-structural Protein 1 of the Virus

Abstract

We developed a Rapid Diagnostic Test (RDT) kit for detecting IgG/IgM antibodies against Zika virus (ZIKV) using monoclonal antibodies to the envelope (E) and non-structural protein 1 (NS1) of ZIKV. These proteins were produced using baculovirus expression vector with Sf9 cells. Monoclonal antibodies J2G7 to NS1 and J5E1 to E protein were selected and conjugated with colloidal gold to produce the Zika IgG/IgM RDT kit (Zika RDT). Comparisons with ELISA, plaque reduction neutralization test (PRNT), and PCR were done to investigate the analytical sensitivity of Zika RDT, which resulted in 100% identical results. Sensitivity and specificity of Zika RDT in a field test was determined using positive and negative samples from Brazil and Korea. The diagnostic accuracy of Zika RDT was fairly high; sensitivity and specificity for IgG was 99.0 and 99.3%, respectively, while for IgM it was 96.7 and 98.7%, respectively. Cross reaction with dengue virus was evaluated using anti-Dengue Mixed Titer Performance Panel (PVD201), in which the Zika RDT showed cross-reactions with DENV in 16.7% and 5.6% in IgG and IgM, respectively. Cross reactions were not observed with West Nile, yellow fever, and hepatitis C virus infected sera. Zika RDT kit is very simple to use, rapid to assay, and very sensitive, and highly specific. Therefore, it would serve as a choice of method for point-of-care diagnosis and large scale surveys of ZIKV infection under clinical or field conditions worldwide in endemic areas.

Keywords: NS1; RDT; Zika; cross reaction; diagnostics; envelope; flavivirus; monoclonal antibody.

Fig. 1

Schematic working principle and the component of ZIKV IgG/IgM RDT kit. Antibody capturing method was applied and 1 more hole for smaller sample application.

Fig. 2

Working procedure of the Zika IgG/IgM RDT kit and the results of the assay. The procedure does not require any specific technic, can be tested easily and rapidly, and showed clear results.

Fig. 3

Purification of the NS1 and envelope proteins of ZIKV proteins and confirmation of the antigenicity. Expressed proteins in Sf9 cells were purified in Ni²⁺-NTA resin (A) and tested for the antigenicity with a ZIKV infected serum in western blot (B).

Fig. 4

Efficacy of the cloned monoclonal antibodies against E and NS1 proteins of ZIKV in RDT kits. Each mAb was conjugated with gold particles and reacted in the test line of the purified ZIKV for selection of useful specific mAb clones.

Fig. 5

Comparison of ZIKV IgG/IgM RDT results with 30 Zika positive sera of confirmed ELISA, PRNT, and PCR results. Real lines showed positive in both IgG and IgM, dotted lines positive only in IgM, and the remainders positive only in IgG.