Tmb single solution

Enzyme-linked immune sorbent assay (ELISA) was used to determine the antibody response levels elicited by E.coli and S2 expressed ZIKV E80 proteins using serum samples obtained from immunized mice, and the cross reactivity of ZIKV immune sera with DENV E80 protein. Briefly, 96-well flat-bottom plates (Costar) were coated overnight with 100μl of diluted ZIKV E80 protein or DENV-3 E80 protein, at a final concentration of 0.5μg/ml. The plates were decanted and then blocked with 5% no-fat milk in PBS containing 0.05% Tween 20 (PBST) for 2 hours at 37°C. Two fold diluted inactivated serum samples were then added in duplicate wells and incubated for 1 hour at 37°C. Horseradish peroxidase (HRP)-conjugated anti-human antibody (Invitrogen) was then added and incubated for 1 hour. Color development was performed using TMB single solution (Life technology), followed by the colorimetric analysis at 450 nm in a 96-well plate reader (Thermo Fisher Scientific).

100 μL of the neutralized reaction samples were transferred to a streptavidin-coated 96-well microtiter plate (Pierce™ Streptavidin Coated High Capacity) and incubated at rt for 1 h. After the incubation, the samples were removed followed by washing of the wells (3 × 300 μL 1 × PBS). 200 μL blocking buffer (2 w/v % skimmed milk protein (Premier Foods) in 1 × PBS (2% MPBS)) was added and allowed to incubate for 1 h at rt or overnight at 4 °C. This blocking buffer was removed followed by a washing step (3 × 300 μL 1 × PBS). 1.0 μg/mL product-specific antibody MGAb (in 2% MPBS) was then added and allowed to incubate for 2 h at rt. After a washing step (3 × 300 μL 1 × PBS), 100 μL polyclonal rabbit anti-mouse IgG-HRP diluted 1:4000 (Invitrogen) in 2% MPBS was added. The plate was incubated at rt for 1 h. Washing with 1 × PBS (3 × 300 μL) primed the plate for the addition of 100 μL TMB single solution (Life Technologies). The TMB solution was allowed to react for 5–15 min in complete darkness by covering with tinfoil. In order to quench the reaction, 50 μL 1 M H₂SO₄ was added, and the plate was analyzed within 10 min of the acid addition at OD₄₅₀ subtracting OD₆₅₅ (EnVision^® plate reader).

For ELISA assay^{38 (link)}, peptides (1.0 μg per well) dissolved in H₂O, ACE2 (100 ng), and chondroitin sulfate (CS, Sigma, Cat^# 230699, 300 ng) or heparan sulfate (HS, Sigma, Cat^# H7640, 300 ng) dissolved in PBS were coated onto ELISA plates and incubated at 4 °C overnight and was blocked at 4 °C overnight. Spike or ACE2 was added to 4H30 wells for binding and then determined by incubation with rabbit anti-spike (Sino, Cat^# 40590-T62, 1:8000) or rabbit anti-ACE2 (Takara, Cat^# A4612, 1:6000), or anti-His-HRP (Invitrogen, Cat^# R93125, 1:2000) at 37 °C for 30 min. 4H30 was added to CS or HS wells for binding and then determined by incubation with rabbit anti-HBD2 (Life Technologies, Cat^# PA5-103126). For determining spike and ACE2 binding, 4H30 or PBST was added to ACE2 for binding and then the spike was added to ACE2 for further incubation at 37 °C for 30 min. The binding ability of spike to ACE2 was determined by incubation with rabbit anti-spike (Sino, Cat^# 40590-T62, 1:8000) at 37 °C for 30 min and then incubation with goat-anti-rabbit HRP (Life Technologies, Cat^#656120, 1:4000) at 37 °C for 30 min. The reaction was developed by adding 100 μL of TMB single solution (Life Technologies, Cat^# 002023) for 15 min at 37 °C and stopped with 50 μL of 1 M H₂SO₄. Readings were obtained in an ELISA plate reader (Victor 1420 Multilabel Counter; PerkinElmer) at 450 nm.

ELISA plates were coated overnight at 4 °C with 100 μL of inactivated PRV HeN1 strain (1 μg/mL) diluted in bicarbonate coating buffer (1.59 g/L Na2CO3 and 2.93 g/L NaHCO3, pH = 9.6). The wells of the plate were washed four times with PBST (PBS containing 0.05% Tween-20) and then blocked with 5.0% skimmed milk in PBST for 2 h at 37 °C. After gradient dilution of mouse serum, 100 μL was added per well, and the plates were incubated for 30 min at 37 °C. The wells were then washed four times with PBST, and 100 μL of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H + L) (Zsbio, Beijing, China) was added at a dilution of 1:5000 for 30 min at 37 °C. Next, plates were washed four times with PBST, and ELISAs were developed using 100 μL of TMB single solution (Life Technologies) for 15 min at 37 °C. The reactions were then terminated with 50 μL/well 2 M H₂SO₄, and the absorbance was measured at 450 nm.

For ELISA assay^{14 (link)}, Peptides (1.0 μg per well) dissolved in H₂O were coated onto ELISA plates and incubated at 4 °C overnight. Then, 2% BSA was used to block plates at 4 °C overnight. For HA and S binding, 150 ng HA1 or S in solution I buffer (Sino Biological Inc., Cat^# 11055-V08H4) was incubated with peptides at 37 °C for 1 h. The binding abilities of peptides to HA1 or S proteins were determined by incubation with rabbit anti-His-HRP (Invitrogen, Cat^# R93125, 1: 2,000) at room temperature for 30 min. The reaction was developed by adding 50 μl of TMB single solution (Life Technologies, Cat^# 002023) for 15 min at 37 °C and stopped with 50 μl of 1 M H₂SO₄. Readings were obtained in an ELISA plate reader (Victor 1420 Multilabel Counter; PerkinElmer) at 450 nm.