Tmb 2 component peroxidase substrate

Maxisorp plates (Nunc) were coated overnight at 4°C with 5 μg of G or F protein per plate in Carb/Bicarb binding buffer (4.41 g KHCO₃ and 0.75 g Na₂CO₃ in 1 L distilled water). After blocking with 5% milk in PBS with 0.01% tween (PBST), serum (2x serial diluted starting at 100x dilution) in 5% milk in PBST was incubated at RT for 1 hr. Antibodies were detected using affinity-purified antibody peroxidase-labeled goat-anti-hamster IgG (Fisher, 14-22-06) in 5% milk in PBST and TMB 2-component peroxidase substrate (Seracare) and read at 450 nm. All wells were washed 3x with PBST in between steps. Prior to using F and G proteins based on NiV Malaysia, we established that cross-reactivity with NiV Bangladesh antibodies was sufficient for usage in ELISA by testing sera known to be positive for NiV Bangladesh antibodies.

Microtiter 96-well flat-bottom medium binding plates (Greiner Bio-One, Kremsmünster, Austria) were coated with recombinant SARS-CoV-2 (WA1 strain) NP (Thermo Fisher Scientific) diluted in 1× DPBS (Corning, Corning, New York, NY, USA) overnight at room temperature. Plates were then washed three times with 1× PBS containing 0.05% Tween 20 (PBS-T). Plates were then blocked for 90 min at 37 °C in 1× PBS 0.5% non-fat milk and 0.1% Tween 20 (blocking buffer). Primary antibodies (mouse sera, hybridoma supernatant, or purified mAb) were diluted in blocking buffer and subsequent serially across the plate. Plates incubated in primary solution for 90 min at room temperature and were then washed three times with blocking buffer. Secondary antibody of either horseradish peroxidase-labeled polyclonal goat anti-mouse IgG antibodies (SouthernBiotech, Birmingham, AL, USA, RRID: AB_2619742) or IgG subclass-specific polyclonal goat anti-mouse antibodies (SouthernBiotech) diluted in blocking were incubated in the wells at room temperature for 60 min. Plates were washed three times with PBS-T. Tetramethylbenzidine (TMB) 2- component peroxidase substrate (SeraCare, Milford, MA, USA) was added to plates and allowed to react for 30 min at room temperature, then stopped using 1 M H₃PO₄. Plates were read at OD 450.

Maxisorp plates (Nunc) were coated overnight at 4 °C with 50 ng/well S or RBD protein in PBS. Plates were blocked with 100 µl of casein in PBS (Thermo Fisher) for 1 hr at RT. Serum diluted 1:6,400 was further 2-fold serially diluted in casein in PBS was incubated at RT for 1 hr. Antibodies were detected using affinity-purified polyclonal antibody peroxidase-labeled goat-anti-hamster IgG (Seracare, 5220-0371) in casein followed by TMB 2-component peroxidase substrate (Seracare, 5120-0047). The reaction was stopped using a stop solution (Seracare, 5150-0021) and read at 450 nm. All wells were washed 4x with PBST 0.1% tween in between steps. The threshold for positivity was set at 2x OD value of negative control (serum obtained from unvaccinated hamsters prior to the start of the experiment).

ELISA was performed as described previously^{26 (link)}. Briefly, maxisorp plates (Nunc) were coated overnight at 4°C with 50 ng/well S or RBD protein in PBS. Plates were blocked with 100 μl of casein in PBS (Thermo Fisher) for 1hr at RT. Serum diluted 1:6,400 was further 2-fold serially diluted in casein in PBS was incubated at RT for 1hr. Antibodies were detected using affinity-purified polyclonal antibody peroxidase-labeled goat-anti-monkey IgG (Seracare, 074-11-021) in casein followed by TMB 2-component peroxidase substrate (Seracare, 5120–0047). The reaction was stopped using stop solution (Seracare, 5150–0021) and read at 450 nm. All wells were washed 4x with PBST 0.1% tween in between steps. Threshold for positivity was set at 2x OD value of negative control (serum obtained from unvaccinated hamsters prior to start of the experiment).

Stabilized SARS-CoV-2 spike protein was obtained from the Vaccine Research Centre, Bethesda, USA. Maxisorp plates (Nunc) were coated overnight at 4°C with 100 ng/well spike protein in PBS. Plates were blocked with 100 μl of casein in PBS (Thermo Fisher) for 1hr at RT. Serum serially diluted 2x in casein in PBS was incubated at RT for 1hr. Antibodies were detected using affinity-purified polyclonal antibody peroxidase-labeled goat-anti-monkey IgG (Seracare, 074–11-021) in casein and TMB 2-component peroxidase substrate (Seracare, 5120–0047), developed for 5–10 min, and reaction was stopped using stop solution (Seracare, 5150–0021) and read at 450 nm. All wells were washed 4x with PBST 0.1% tween in between steps. Threshold for positivity was set at 3x OD value of negative control (serum obtained from non-human primates prior to start of the experiment) or 0.2, whichever one was higher.