Tbs blocking solution

Cohesin fusion proteins were expressed in CHO-S cells from expression vectors containing the HIV-1 Env gp140 sequence (16 (link)) or Env fragments (gp120 residues 94–1550; gp41 residues 1551–2064, inserted between the Cohesin domain and 6 C-terminal His codons). ELISA plates were coated overnight at 4°C with 2 μg/ml of the Cohesin fusion proteins in 0.2 M sodium carbonate-bicarbonate buffer, pH 9.4. Serial dilutions of serum starting at 1:500 in TBS blocking solution (StartingBlock T20, Pierce or Thermo Fisher Scientific) were incubated in the wells overnight at 4°C. After washing, plates were incubated with HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) in TBS blocking solution (Thermo Scientific, Rockford, IL, USA) for 2 h at 37°C, then washed and developed with HRP substrate (TMB, Life Technologies), stopped with equal volume of 1N HCl and read at 450 nm. The log10 transformed and normalized areas under the curves (AUC) data were calculated for each animal at each time point using GraphPad Prism 8 software. EC50 calculations were based on log10 transformed and normalized data with non-linear regression curve fit using sigmoidal dose response with variable slope constraints.

Cohesin fusion proteins [30 (link)] were expressed in CHO-S cells from vectors containing the above Env gp140 sequence, or Env fragments (gp120 residues 94–1550; gp41 residues 1551–2064, inserted between the cohesin domain and 6 C-terminal His codons,). HIV Env-specific IgG antibody titers in serum from vaccinated animals were assessed at indicated time points post-immunization using ELISA. ELISA plates were coated with 2 μg/ml of the cohesin fusion proteins in 0.2 M sodium carbonate-bicarbonate buffer, pH 9.4. Serial dilutions of serum starting at 1:500 in TBS blocking solution (StartingBlock T20, Pierce) were incubated in the wells overnight at 4°C. After washing, plates were incubated with HRP-conjugated goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) in TBS blocking solution (Thermo Scientific, Rockford, IL) for 2 h at 37°C, then washed and developed with HRP substrate (TMB, Life Technologies), stopped with equal volume of 1N HCl and read at 450 nm. The log₁₀ transformed and normalized areas under the curves (AUC) data were calculated for each animal at each time point using GraphPad Prism 6 software (GraphPad, La Jolla, CA). EC50 calculations were based on Log10 transformed and normalized data with non-linear regression curve fit using sigmoidal dose response with variable slope constraints.