Hrp conjugated goat anti human igg secondary antibody

Serum binding antibody titers against clade C gp140 trimer were determined by endpoint ELISAs as previously described [11 (link), 12 (link)]. Briefly, 96-well Maxisorp ELISA plates (Thermo Fisher Scientific), coated over-night with 100 μl/well of 1 μg/ml clade C gp140, were blocked for 2.5 hours with PBS/Casein (Pierce). Guinea pig sera were then added in serial dilutions and incubated for 1 hour at room temperature. The plates were washed three times with PBS containing 0.05% Tween 20 (Sigma) and incubated for 1 hour with a 1/2,000 dilution of a horseradish peroxidase (HRP)-conjugated goat anti-guinea pig secondary antibody (Jackson ImmunoResearch Laboratories) or HRP-conjugated goat anti-human IgG secondary antibody (Fisher Scientific) for NHP assays. The plates were washed three times and developed with SureBlue tetramethylbenzidine (TMB) microwell peroxidase (KPL Research Products), stopped by the addition of stop solution (KPL Research products), and analyzed at 450 nm/550 nm with a Spectramax Plus ELISA plate reader (Molecular Devices) using Softmax Pro 4.7.1 software. ELISA endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance >2-fold over background values.

PGDM1400 and CAP256-VRC26.25-LS antibody concentration in serum was determined by ELISA as described (41 (link)) using the BG505 SOSIP trimer or the novel CNE58-strandC-CAP256.SU SOSIP trimer, respectively. Briefly, microtiter plates were coated with 1 μg/mL SOSIP trimer and incubated overnight at 4°C. The plates were washed with PBS/0.05% Tween-20 and blocked with PBS/Casein (Pierce). After blocking, serial dilution of serum samples were added to the plate and incubated for 2 h at 37 °C. Binding was detected with a horseradish peroxidase (HRP)-conjugated goat anti-human IgG secondary antibody (Fisher Scientific) and visualized with SureBlue tetramethylbenzidine (TMB) microwell peroxidase (KPL Research Products).