Peroxidase conjugated goat anti human igg

Huh7 cells were infected with DENV with or without resveratrol (RESV) (Sigma-Aldrich Chemical) added to cells at various concentrations. Viral titers in culture supernatants were determined as plaque- or focus-forming units (PFU or FFU)/ml. For this, 1 × 10⁵ BHK or Vero cells per well in 12 or 24-well plates (Costar, Corning, NY) were inoculated with serially diluted supernatants (lowest dilution 1:2). The plaque assay was performed and stained with crystal violet (Sigma-Aldrich Chemical). For FFU assay, after infection for 72 h, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min and permeabilized with 1% Triton X-100 in PBS for 15 min at room temperature (RT). After three washes with PBS, fixed cells were blocked with 3% skimmed milk in PBS for 2 h at RT. Cells were then stained with primary antibody (1:500 of human serum) and secondary antibody (1:250 of peroxidase-conjugated goat anti-human-IgG; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at 37 °C. The immunostained cells were visualized with a 3, 3′-diaminobenzidine (DAB) substrate (Vector Laboratories, Burlingame, CA). FFUs were counted and infectivity titers were calculated.

Serum IgG antibodies against SARS-CoV-2 Spike protein were measured by an ELISA developed and standardized in our laboratory. Briefly, 96-well microplates (Immulon 4HBX, Thermo Scientific) were coated with recombinant NCP-CoV (2019-nCoV) Spike protein (S1+S2 ECD) (Sino Biological #40589-V08B1) at 2 μg/mL for 1 hr at room temperature. Plates were then washed with Tween-20 0.05% in PBS (PBST) and blocked with assay buffer (1% BSA in PBS) for 1 hr at 37°C. After blocking, all subsequent steps were performed by a DYNEX DS2® Automated ELISA system (DYNEX Technologies, Chantilly, VA, USA). Serum samples diluted 1:50,000 in assay buffer were added in duplicate, and plates were incubated for 2 hrs. Plates were washed with PBST and 100 μL per well of a peroxidase-conjugated goat anti-human IgG (Jackson ImmunoResearch #109-036-098), diluted 1:10,000 in assay buffer, were added. After 1 hr incubation, plates were washed, and a stabilized 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Sigma) was added to the wells. The enzymatic reaction was stopped after 20 min with a Stop solution (1 M sulfuric acid), and absorbance at 450 nm was read by the DYNEX DS2 instrument.

Liposomes were diluted in ethanol to a final concentration of 25 µM phospholipids and coated on Immuno MaxiSorp plates (NUNC, Roskilde, Denmark). 1% BSA (BSA; Fraction V, Fatty acid free, Calbiochem, San Diego, CA, USA) diluted in PBS was used to block coated plates. Next, samples were incubated with CD169 Fc or its mutant form (CD169 Fc R97A) (2 μg/mL) for 1 hour at room temperature (kindly provided by Prof. Dr. P.R. Crocker, University of Dundee) (57 (link)). Then, peroxidase-conjugated goat anti-human IgG (Jackson ImmunoResearch, Ely, UK) was added for an additional 30 minutes and plates were washed, TMB (Sigma Aldrich, Darmstadt, Germany) was added as a substrate and the optical density (OD) measured in a microplate absorbance spectrophotometer (Biorad, Hercules, CA, USA) at 450 nm.