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

Kim, Yeong Hoon; Lee, Jihoo; Kim, Young-Eun; Chong, Chom-Kyu; Pinchemel, Yanaihara; Reisdörfer, Francis; Coelho, Joyce Brito; Dias, Ronaldo Ferreira; Bae, Pan Kee; Gusmão, Zuinara Pereira Maia; Ahn, Hye-Jin; Nam, Ho-Woo

We produced recombinant NS1 and E proteins of ZIKV through recombinant baculovirus expression vector technology with Sf9 cells. The pAcGP67a-NS1-his and pAcGP67a-Envhis vectors were cloned. Briefly, Spodoptera frugiperda Sf9 cells (ATCC #CRL-1711) were grown at 27ËšC in Sf-900II serum-free medium (Gibco/BRL, Gaithersburg, Maryland, USA) with 10% fetal bovine serum (FBS). The Sf9 cells were transfected with pAcGP67a-NS1-his and pAcGP67a-Env-his vector by polyfect-mediated method (Hilden, Qiagen, Germany). The cells were incubated at 27ËšC for 72 hr, and then infected with recombinant baculoviruses at a multiplicity of infection (MOI) 0.01-10. Following 7 days of incubation at 27ËšC the cells were removed from culture medium. The supernatant was collected and analyzed on SDS-PAGE and western blot. With the cold centrifugation at 3,000 g for 10 min, the collected supernatant was introduced to pre-equilibrated Ni 2+ -nitrilotriacetic acid (Ni-NTA) resin (Qiagen). Elution was performed using 5 and 500 mmol/L imidazole in 20 mmol/L Tris-Cl (pH 8.0). The eluate was dialyzed using Tris-Cl (pH 8.0) for 24 hr by changing the buffer thrice. Western blot against Zika positive and anti-hIgG-HRP, and SDS-PAGE were performed after purification.