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Human IgGs against WA-1 SARS-CoV-2 S RBD and N were quantified using ELISA as described (54 (link)). WA-1 S RBD (2 μg/ml) or N (1 μg/ml) proteins were coated on plates and blocked with 3% milk in 0.1% Tween 20 +PBS (PBS-T). Plates were washed three times with PBS-T (200 μl) and virus inactivated samples (25 μl) from human plasma or infected mice were added to 100 μl of sample buffer (1% milk + 0.1% Tween 20 diluted in PBS) and serially diluted (5-fold) down the plates. The final row was left with 100 μl of sample buffer as a negative control. Plates were incubated for 10 min at room temperature shaking at 60 rpm and subsequently washed four times with PBS-T (200 μl). Secondary antibody (100 μl 1:500 anti-human IgG HRP, Invitrogen 31410) was added and plates were incubated for 10 min at room temperature shaking at 60 rpm. After incubation, plates were washed five times with PBS-T (200 μl) and SigmaFAST OPD (Sigma-Aldrich P9187, 100 μl) was added to each well of the plate. OPD development was stopped with 25 μl of 3 M hydrochloric acid and plates were read at an absorbance of 492 nm on a Synergy H1 plate-reader. Binding antibody units (BAU) were calculated based on the NIBSC 1st WHO International Standard (NIBSC code 20/136). Area under the curve analysis was completed in GraphPad Prism v.9.

Humoral immune responses directed against each antigen were monitored for each animal. Ninety six-well plates (Polysorp, Nunc) were coated with 0.5 μg recombinant protein per well and incubated for one hour at 37 °C. All washing steps were carried out using Phosphate Buffered Saline (10 mM PO₄³⁻, 137 mMNaCl and 2.7 mM KCl) containing 0.5 % (v/v) Tween 20 (PBST). Plates were washed three times in an automated plate washer for each wash step. Plates were blocked with 1 % casein in PBST for 30 min at 37 °C. Serum was applied at an initial concentration of 1:500 followed by 1:2 dilutions and incubated for one hour at 37 °C. After washing, anti-bovine IgG, whole molecule, peroxidase conjugate (Sigma, St. Louis, USA) was added at a dilution of 1:10,000 in PBST and incubated for one hour at 37 °C. Plates were washed and given a final rinse in PBS. Plates were developed using SIGMAFAST OPD (Sigma, St. Louis, USA) following instructions and signals were evaluated at optical density (OD) 450 nm in a plate reader. Mean readings (with standard error) were calculated for each time point by grouping control and vaccinated animals. Endpoint titres were determined as the last serum dilution where the OD of test sera was ≥ 2 OD of negative control bovine serum donor. Time points evaluated were before vaccination and two weeks after each inoculation (days 0, 14, 42 and 70).

Pooled, heat-inactivated, and RDE-treated sera (starting concentration 1:50 and serially diluted 2-fold) and viruses (1000 PFUs) were preincubated at room temperature for 1 h to allow antibodies to bind to virions.^{42 (link)} After incubation, the mixture was added to monolayers of MDCK cells in 96-well tissue culture plates and incubated at 37 °C for 1 h to allow for attachment of virions to the cells. After washing with PBS three times to remove non-attached virions, the plates were re-incubated at 37 °C with infection medium containing the appropriate serum dilution. Eighteen hours later, the cells were fixed with 80% acetone in PBS and then stained for the NP protein using a primary biotinylated antibody (EMD Millipore; Cat. no. MAB8257B) (1:2000) and a secondary streptavidin conjugated to HRP (EMD Millipore; Cat. no. 21130) (1:5000). Wells were developed by incubating with SigmaFast OPD (Sigma-Aldrich) for 20 min. Reactions were stopped by adding 3 M HCl and absorbance at 490 nm was determined on a Synergy 4 plate reader (BioTek). Endpoint titers were defined as the reciprocal of the highest serum dilution that neutralized virus.

Plasma was collected from heparinized whole blood and stored at −80°C until use. HCMV infection status was determined by HCMV IgG ELISA (BioKit). IgG Abs to pertussis toxin (PT; NIBSC) and to formalin-inactivated whole H1N1 influenza virus (influenza A/California/7/2006(H1N1)v(NYMC-X179A); H1N1; NIBSC) were determined using in-house ELISA assays with goat anti-human IgG-peroxidase (Sigma-Aldrich) as the secondary Ab and SIGMAFAST OPD (Sigma-Aldrich) as the substrate. IgG concentrations were calculated by interpolation from a standard curve, which was produced using anti-pertussis reference serum (NIBSC; IU/ml) or using plasma from a donor with high titers of Abs to H1N1 influenza (IgG concentration expressed in arbitrary ELISA units [AEU]) (28 (link)). The pooled AB plasma used for in vitro assays contained 6.8 IU/ml IgG to PT and had an H1N1 IgG titer of 273.8 AEU.