ZIKV Envelope Protein Detection Assay

Monolayers of BHK-21 cells (ATCC, USA) were transfected with the constructs using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. After 48h, cells were fixed using 80% acetone for 1h at room temperature (RT) and blocked with assay buffer containing 5% skimmed milk (Bio-Rad, USA) in PBS-T buffer (1X PBS with 0.05% Tween-20) for 1h at RT. For ZIKV E detection, the 4G2 monoclonal antibody (34 (link)) (1μg/mL) was added in assay buffer for 2h at 37°C. Plates were washed five times with PBS-T, followed by peroxidase-conjugated anti-mouse IgG antibody (Sigma Aldrich, USA) for 1h at RT. After a final wash step, the reaction was revealed with tetramethylbenzidine (TMB, KPL SureBlue Reserve, USA) substrate for 45 min at RT, stopped with 1N HCl and read at 450nm (OD 450nm) using a microplate spectrophotometer (Benchmark Plus, Bio-rad, USA). A plasmid coding for YFV proteins (pL/YFV) was used as a positive control (18 (link)). All samples were tested in duplicate, and the results were considered valid when intra-assay variability was below 20%.