Goat anti human igg conjugated with hrp

Wells of microtiter plates were coated with purified, recombinant EBOV, BDBV, SUDV, or MARV GP ΔTM and incubated at 4°C overnight. Plates were blocked with 2% non-fat dry milk and 2% normal goat serum in DPBS containing 0.05% Tween-20 (DPBS-T) for 1 hr. For mAb screening assays, hybridoma culture supernatants were diluted in blocking buffer 1:5, added to the wells, and incubated for 1 hr at ambient temperature. The bound antibodies were detected using goat anti-human IgG conjugated with HRP (Southern Biotech) and TMB substrate (ThermoFisher). Color development was monitored, 1N hydrochloric acid was added to stop the reaction, and the absorbance was measured at 450 nm using a spectrophotometer (Biotek).
For dose-response and cross-reactivity assays, serial dilutions of plasma or purified mAbs were applied to the wells in triplicate or quadruplicate, as detailed above. EC₅₀ values for mAb binding were determined using Prism 7.2 software (GraphPad) after log transformation of antibody concentration using sigmoidal dose-response nonlinear regression analysis. Similarly, a non-linear regression analysis was performed on the resulting curves to calculate plasma dilution that yielded a half-maximum O.D. 450 nm value. Antibody titer in plasma was expressed as the inverse of plasma dilution.

ELISA plates were coated with S1 protein at 50 mM in carbonate coating buffer (pH = 9.6) at 4 °C overnight. After standard washing and blocking, diluted sera (from 1:100 to 1:30000) were applied to each well. After a 1 h incubation at 37 °C, plates were washed and incubated with 0.25 μg/ml goat anti-human IgG-conjugated with HRP (Southern Biotech, Birmingham, AL, USA) for 1 h at 37 °C. TMB was used as the substrate, and the reaction was ceased by 2 mol/L H₂SO₄. A microplate reader measured the absorbance at 450 nm.

Wells of microtiter plates were coated with purified, recombinant EBOV, BDBV, SUDV, or MARV GP ΔTM and incubated at 4 °C overnight. Plates were blocked with 2% non-fat dry milk and 2% normal goat serum in DPBS containing 0.05% Tween-20 (DPBS-T) for 1 h. Serial dilutions of mAb were applied to the wells and incubated for 1 h at ambient temperature. The bound antibodies were detected using goat anti-human IgG conjugated with HRP (diluted 1:5000) (Southern Biotech, CAT: 2040–05) and TMB substrate (ThermoFisher Scientific). Color development was monitored, 1 N hydrochloric acid was added to stop the reaction, and the absorbance was measured at 450 nm using a spectrophotometer (Biotek). EC₅₀ values for mAb binding were determined using Prism 7.2 software (GraphPad) after log transformation of antibody concentration using sigmoidal dose-response nonlinear regression analysis.