Hrp conjugated goat anti human igg

For detection of antibody binding to HIV virus-like particles (Env VLP or Gag-only VLP) or to monomeric recombinant HIV gp120 protein, Immulon II HB 96-well microplates (Dynex Technologies) were coated with 100 μL of Env VLP, Gag-only VLP diluted 1:200 in D-PBS, or monomeric gp120 protein diluted 1:5,000 in PBS, and stored overnight at room temperature. The plates were blocked for 2 h at 4°C with 200 μL per well of D-PBS containing 10% (v/v) FBS. We added 100 μL of antibody at a concentration of 1 μg/mL in diluent (7.5% FBS, 0.05% Tween-20 in D-PBS) to individual wells, incubated the plate for 1 h at 37°C, washed, and detected binding with 100 μL of HRP-conjugated goat anti-human IgG (Southern Biotechnology Associates) diluted 1:1,000 in diluent. The plates were washed, and 100 μL of TMB substrate (Pierce) was added to individual wells, and 50 μL of 1N HCL was added to each well to stop the reaction. The optical density of solutions in the plates was read at 450 nm using a Spectramax M5 plate reader (Molecular Devices).

For testing bivalent Fabs, 96-well Maxisorp ELISA plates (Thermo Scientific) were coated with RSV prefusion or postfusion F protein overnight at 4°C. Threefold serially diluted antibodies were prepared in 1% nonfat milk/TBST, transferred to antigen coated plates, and incubated for 1 hr at RT with shaking at 150–200 rpm. Plates were washed 5 times with TBST. 100 μL per well of 1:40,000 diluted alkaline phosphatase-conjugated goat anti-human IgG ((Fab’)₂ specific, Jackson ImmunResponse) was then added and incubate for 1 hr at room temperature with shaking at 150–200 rpm. Plates were washed 5 times with TBST. 100 μL of AttoPhos substrate was added to each well (1:5 diluted in TBST). After 4-7min incubation at RT, fluorescence signals were read with a Tecan F500 plate reader at 435 nm for excitation and 530 nm for emission. For testing full-length human IgGs, 1:2,000 diluted HRP-conjugated goat anti-human IgG (Southern Biotech) was used as secondary antibody. SuperBlu-Turbo TMB substrate (ViroLABS) was used as substrate for color development and plates were read at 450 nm.

Briefly, 96-well high-binding EIA/RIA plates (Costar) were coated with 50 μL/well of 1 ug/mL spike or NP proteins in phosphate-buffered saline (PBS) overnight. The coated plates were washed twice with 200 μL/well of PBS containing Tween 20 (PBS-T), blocked with blocking buffer (1% blotting-grade blocker (BIO-RAD) in PBS-T) for 30 min at 37 °C. Plasma samples were serially diluted 4-folds in blocking buffer, added to the plates, and incubated at room temperature (RT) for 2 h. The plates were washed three times with PBS-T, and HRP-conjugated goat anti-human IgG (SouthernBiotech) in blocking buffer was added, followed by incubation for 1.5 h at RT. After washing three times with PBS-T, the wells were treated with TMB substrate solution (OptEIA reagent set, BD). Finally, the reaction was stopped by the addition of a stop solution (0.5 M hydrochloric acid). Optical density at 450 nm was measured using a microplate reader (Victor 3, PerkinElmer).

Maxisorp 384 well plates (Nunc, #P6366) were coated with 1 µg/mL of Wuhan NTD (amino acids 1 to 162 of spike) or trimeric Wuhan spike proteins prepared as described previously^{70 (link)} at 4 °C overnight. After washing with wash buffer (PBS supplemented with 0.05% Tween-20), the plates were blocked with 1% BSA (Roche, #10735086001)/PBS at room temperature for 1 h. After washing with the wash buffer, the plates were incubated with serially diluted plasma (6-fold serial dilutions starting from 1:500) in Diluent 100 (Meso Scale Discovery) for 2 h at room temperature. The plates were washed with the wash buffer and incubated with HRP-conjugated goat anti-human IgG (1:5000, polyclonal, Southern Biotech, #2040-05) for 1 h at room temperature. After washing with the washing buffer, HRP activity was visualized with OPD substrate (Sigma, #523121), followed by the addition of HCl to stop the reaction. Absorbance at 490 nm was measured using Epoch2 (Biotek). Binding curves were analyzed with GraphPad Prism software with 4-parameter nonlinear regression to calculate the EC₅₀ values.