Casein tbs

We adapted an ELISA protocol as previously described (Derking et al., 2015 (link)). In brief, His-tagged trimers, either pure (3.5 µg/ml in TBS buffer) or in unpurified HEK293T cell culture supernatant (His- and D7324-tagged; supplemental information), were immobilized (100 µl/well) for 2 h on 96-well Ni-NTA ELISA plates (QIAGEN) or 96-well ELISA plates coated overnight with D7324 antibody (Aalto Bioreagents). After washing away excess protein with TBS, the wells were blocked for 30 min with casein/TBS (37532; Thermo Fisher Scientific). Serial dilutions of each antibody were prepared in casein/TBS at a starting concentration of 1 µg/ml and added to the plate (100 µl/well; for lower affinity antibodies, the starting concentration was 50 µg/ml). The dilution factor for all antibodies was 1:3 except for gl-CH103, which was 1:2. Excess antibody was washed away after 2 h and antihuman HRP-conjugated antibody (diluted in casein/TBS 1:3,000) added for 45 min before binding was quantified. All steps were performed at room temperature.

A 1 µg mL⁻¹ concentration of His-tagged SOSIP protein in Tris-buffered saline (TBS) was used to coat wells of a 96-well Ni-NTA ELISA plate for 2 h (Qiagen). After three washes with TBS, the wells were blocked for 30 min with casein/TBS (ThermoFisher Scientific). A 1 µg mL⁻¹ concentration of antibody in casein/TBS were serially diluted 1:3 and incubated at room temperature (RT). Plates were washed after 2 h with TBS and a 1:3000 dilution of goat anti-human horseradish peroxidase (HRP)-conjugated antibody (109-035-008; Jackson Immunoresearch) in casein/TBS was added for 45 min before binding was quantified. Next, after washing the plates five times with TBS + 0.05% Tween-20, developing solution (1% 3,3’,5,5’-tetranethylbenzidine (Sigma-Aldrich), 0.01% H₂O₂, 100 mM sodium acetate, and 100 mM citric acid) was added. Development of the colorimetric endpoint proceeded for 3 min before termination by adding 0.8 M H₂SO₄.