Tetramethylbenzidine tmb substrate

For the evaluation of the virus-specific antibodies levels present in the sera of vaccinated mice, enzyme-linked immunosorbent assays (ELISAs) were performed as previously described [45 (link)]. Briefly, 96-well plates were coated with cell lysates from mock- or ZIKV-infected Vero cells and incubated overnight at 4 °C. The coated wells were washed with PBS, blocked with 1% BSA in PBS, and then incubated with two-fold dilutions (starting dilution of 1:50) of mice sera for 1 h at 37 °C. After that, plates were washed with water and incubated with HRP-conjugated goat anti-mouse IgG (1:2000; Southern Biotech, Birmingham, AL, USA) for 1 h at 37 °C. Reactions were developed with tetramethylbenzidine (TMB) substrate (BioLegend, San Diego, CA, USA) for 10 min at room temperature, quenched with 2 N H₂SO₄, and read at 450 nm in a Vmax Kinetic microplate reader (Molecular devices, San Jose, CA, USA).

Blood was collected by cardiac puncture at post-mortem (PM) examination and serum was stored at − 80 °C following centrifugation of clotted blood at 1000 g for 10 min at 4 °C. Serum IgY levels were quantified by ELISA as described. To quantify vaccine-specific responses, 96-well plates were coated with 0.1 μg/ml of purified unG-NetB or G-NetB(10) antigens in carbonate-bicarbonate buffer and incubated at 4 °C overnight. Plates were washed three times with PBS containing 0.05% (v/v) Tween 20 and 50 μl of 1:50 serum diluted in PBS was added per well. Control wells were maintained to which no serum was added. Plates were incubated at room temperature for 1 h and then washed as above. Rabbit anti-chicken IgY-horseradish peroxidase (HRP) antibody at 1:3000 (Sigma, UK) was used to detect bound serum IgY. Plates were washed three times, tetramethylbenzidine (TMB) substrate (BioLegend, UK) was added and the plates were incubated for 10 min at room temperature in the dark. The reaction was stopped using 2 M H₂SO₄ and optical density at 450 nm adjusted against optical density at 620 nm (OD_450/620 nm) was measured using a plate reader (Biotek Cytation 3) with background correction using the values of the no serum control wells.