Anti 6x his tag antibody conjugated to horseradish peroxidase hrp

The capacity of the covNHR proteins to bind soluble HIV-1 envelope proteins (Env) was determined by ELISA. Briefly, 96-well ELISA plates (Maxisorp, Nunc) were coated at 4 °C overnight with various Envs (Table S1) in 0.1 M bicarbonate buffer (pH 9.6). After saturation with 2% BSA, 0.05% Tween in PBS for 1.5 h at 25 °C, 0.01 μM of covNHR molecules, corresponding to the protein concentrations that allowed detecting optical density changes within a linear range, (100 μL diluted in 1% BSA 0.05% Tween solution) were added and incubated for 2 h at room temperature. The plate was then washed five times and covNHR binding was detected with 100 μL anti-6X His-tag antibody conjugated to horseradish peroxidase (HRP) (Abcam, Cambridge, UK) at 1/dilution incubated for 1 h at room temperature. Antibody binding was then revealed with tetramethylbenzidine (TMB) substrate buffer, the reaction was stopped with 1 M H₂SO₄ and optical density was read at 450 nm with a Molecular Device Plate Reader equipped with SoftMax Pro 6 program. Background binding was measured in plates without Env and subtracted from the data. The percentage of binding was calculated using the readings with wells coated with His-tagged Env incubated with PBS buffer instead of covNHR molecules as a control for 100% binding.