Ni nta elisa plates

To assess the binding of serum antibodies from weeks 0, 4, 6, 12, 22, 36, and 38 against the AMC009, AMC011, and AMC008 SOSIP trimers, the purified His-tagged trimers were immobilized on Ni-NTA ELISA plates (Qiagen) (80 (link)). Subsequent steps are the same as those for D7324 capture ELISA.

For D7324-capture ELISA, supernatants from HEK293T cells containing unpurified SOSIP trimers, or PGT145-purified SOSIP trimers (1.0 µg/mL), were diluted in Tris-buffered saline (TBS). The trimers were then immobilized via their D7324-tags for 2 h at room temperature on half-well 96-well plates (Greiner) precoated with Ab D7324 (Aalto Bioreagents) at 10 µg/mL in 0.1 M NaHCO₃ pH 8.6 overnight^{4 (link),50 (link)}. Midpoint and end point antibody titers from sera samples were determined in D7324-capture ELISA as previously described^{3 (link)}. For lectin-capture ELISA, half-well 96-well plates were coated with Galanthus nivalis lectin (Vector Laboratories) at 20 µg/mL in 0.1 M NaHCO₃ pH 8.6 overnight, which were then blocked using Casein Blocker (Thermo Scientific) followed by immobilization of the purified SOSIP trimer and SOSIP-ferritin proteins in TBS (2.0 µg/mL). Subsequent steps were performed as previously described^{4 (link)}. For Ni-NTA His-capture ELISA, the purified SOSIP trimers (1.0 µg/mL) were diluted in TBS and immobilized on 96-well Ni-NTA ELISA plates (Qiagen). Subsequent steps were performed identical to the D7324-capture ELISA. For the ELISA in Supplementary Fig. 9, 2 µg/mL of purified ferritin cages were coated overnight on half-well 96-well plates, which were then blocked using Casein Blocker. Subsequent steps were performed as described above.

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.