Blocker casein

Ten µl of each sample were loaded onto 7.5%, 12% Tris-glycine SDS-PAGE gels or 4–20% gradient gels (Biorad) depending on the molecular weight of the proteins of interest. Proteins were transferred to PVDF membranes and subsequently blocked with casein blocker (Thermo Scientific) or non-fat milk (Cell Signalling Technologies) for 1 hr at room temperature. Membranes were probed with primary antibody overnight at 4 °C and following washing steps were incubated with the appropriate HRP-conjugated secondary antibody for 2 hr at room temperature. The signal was detected using a CCD camera and Luminata Crescendo (Merck Millipore) or West Femto (Pierce) chemiluminescent HRP substrate depending on the intensity of the signal.

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.

Maxisorp plates (Nunc-Immuno) were coated at 4 °C overnight in bicarbonate buffer with the anti-PfRH5 mouse mAb 2AC723 (link) at 5 μg/mL. Plates were washed with PBS/0.05% Tween-20 (PBS/T), blocked in Casein Blocker in PBS (Thermo Scientific) for 1 h at room temperature (RT), and washed again prior to addition of diluted PfRh5 protein samples in duplicate. Serially diluted pure PfRH5 v1.0 in the range 200 ng/mL to 1.56 ng/mL was added to each plate to generate a standard curve. Plates were incubated at RT for 2 h and then washed prior to addition of rabbit serum from animals immunized with PfRH5 (3D7) viral vectored vaccine7 (link) diluted at 1:1000 in Casein Blocker. After a further 1 h incubation and subsequent wash, alkaline phosphatase-labelled goat anti-rabbit IgG (whole molecule) (Sigma) was added to each well at a 1:5000 dilution in Casein Blocker and plates were left to incubate for a final hour. Bound antibodies were detected by addition of p-nitrophenyl phosphate substrate (Sigma) diluted in diethanolamine buffer (Thermo Scientific). The optical density at 405 nm (OD₄₀₅) was read using an ELx800 microplate reader (BioTek, UK) and a four parameter fit was generated from standard curve values with Gen5 ELISA software v1.10 (BioTek, UK). This curve was used to convert the absorbance of diluted PfRH5 samples into concentration of PfRH5 protein.

For assessing the thermostability of 2G12 and PGT145 epitopes on the different SOSIP proteins, we submitted the trimers for 30 min to a set of temperatures ranging from 25 °C to 90.6 °C. Subsequently, they were diluted in TBS and immobilized on Ni-NTA as above described. Antibody binding was assessed by incubation with a single Casein Blocker (Thermo Scientific) solution with a 2G12 or PGT145 concentration close to their priorly determined IC₅₀ (0.1 µg/mL). Subsequent steps to assess the binding of the test antibodies were performed as above described. Temperatures of melting (T_m) were determined by interpolation of the temperature values that corresponded to 50% of the maximum binding signal, using Graphpad Prism 8.4.3

Soluble antigen was diluted to 2 μg/mL in Carbonate Coating Buffer (0.2 M Carbonate-Bicarbonate, pH 9.4 (Thermo Fisher)) and 20 μL was dispensed into the wells of a 384-well High Binding microplate (Greiner). The plate was sealed and incubated at 4°C overnight. After washing 3X with PBS + 0.1% Tween-20 (in house), the plate was blocked with 80 μL of Casein Blocker in PBS (Thermo Fisher) for 60 minutes. Following washing, 20 μL of normalized CHO supernatants at 5, 1.25, 0.31 and 0.08 μg/mL were added to the wells of the ELISA plate and incubated for 60 minutes. The expression supernatants derived from minipreps for both the standard primers and the rh-PCR Generation 2 primers were tested. After washing 3X with PBS + 0.1% Tween-20, 20 μL of 1:1000 dilution of Goat anti-Human kappa-AP (Southern Biotech) in Casein Blocker was added to each well and incubated 60 minutes. After washing again, 20 μL of 1-Step PNPP (Thermo Fisher) was added and the plate incubated until color developed. The plate was read on a Spectramax 384 Plus spectrophotometer at 405 nm.