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

PRNT was performed to determine the presence of virusspecific neutralizing antibodies in paired serum samples. Briefly, Vero cells were seeded at a density of 160,000 cells/well 24 hr before infection in 6-well plates. De-complemented sera samples (30 min at 56˚C) were 2 fold serially diluted from 1: 10 to 1:400 in DMEM serum-free medium. Then an equal volume of DMEM-diluted ZIKA virus containing 200 PFU was added and incubated for 1 hr at 36˚C in a final volume of 200 μl. Vero cells were infected in duplicate with 100 μl of the neutralization mixture and incubated for 1 hr at 37˚C. Afterwards, the viral inoculum was removed and cells were overlaid with 2 ml of DMEM with 2% FBS and 1.5% SeaPlaque agarose (Lonza). Plates were incubated at 37˚C for 3-5 days. After this period, cells were washed twice with PBS, stained with 1% crystal violet for 30 min. Plaques were counted and percentage of plaque reduction against control serum was calculated.