SDS-PAGE: Coomassie-stained SDS-PAGE showing Cytomegalovirus gH Pentamer Complex.
Direct ELISA: Plate was coated with 1000ng, 100ng, 10ng or 1ng of NRP-2 and probed with 500µg per well of NRP-2, gH pentamer or gB huFc-tag and then blocked for 1 h at room temperature. Plate was probed with 500µg per well of NRP-2, gH pentamer or gB huFc-tag for 2h at room temperature. For primary antibodies, sheep anti-gH pentamer serum was used at 1:2,000, mouse anti-His-tag antibody was used at 1:1,200 (for ZIKV-NS1), no primary antibody was used for gB. Secondary antibody STAR88P was used for gH pentamer at 1:2,000; STAR147P was used for gB huFc-tag detection via Fc-tag at 1:2,000 and Biorad 105300 was used to detect ZIKV-NS1 at 1:2,000. All secondary incubations were done for 45 min at room temperature. Detection was carried out using TMB Membrane substrate (KPL) for 40 min. 1M HCl was used to stop the reaction. Assay confimred that NRP-2 binds specifically to gH pentamer but not to gB or ZIKV NS1.