To screen hybridomas, micro-ELISA plates (Nunc, USA) were coated overnight at 4 °C with 200 ng/well of ZIKV E80 protein, and blocked with 5% milk in PBS-Tween20 (PBST). 50 μL of undiluted hybridoma culture supernatants was added to the plates and incubated at 37 °C for 2 h. Plates were then washed with PBST and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Sigma, USA). After washes and color development, absorbance at 450 nm was measured.
Immunoglobulin isotypes of the mAbs were measured using SBA ClonotypingTM System/HRP ELISA kit (Southern Biotech, USA) according to manufacturer’s instructions.
To measure binding properties of these mAbs, microplates (Nunc) were coated at 4 °C overnight with 200 ng/well of ZIKV E80, or DENV2 E80, and then blocked with 5% milk in PBST. Next, 50 μL/well of serially diluted anti-ZIKV mAbs, anti-EV71 mAb D5 (isotype control)35 (link) or anti-DENV mAb D1-11 (Santa Cruz Biotechnology, USA) were added and incubated at 37 °C for 2 h. After washing with PBST, plates were incubated with HRP-conjugated anti-mouse IgG (Sigma). After color development, absorbance at 450 nm was measured.
Immunoglobulin isotypes of the mAbs were measured using SBA ClonotypingTM System/HRP ELISA kit (Southern Biotech, USA) according to manufacturer’s instructions.
To measure binding properties of these mAbs, microplates (Nunc) were coated at 4 °C overnight with 200 ng/well of ZIKV E80, or DENV2 E80, and then blocked with 5% milk in PBST. Next, 50 μL/well of serially diluted anti-ZIKV mAbs, anti-EV71 mAb D5 (isotype control)35 (link) or anti-DENV mAb D1-11 (Santa Cruz Biotechnology, USA) were added and incubated at 37 °C for 2 h. After washing with PBST, plates were incubated with HRP-conjugated anti-mouse IgG (Sigma). After color development, absorbance at 450 nm was measured.