Data analysis 11

To evaluate the antigen-binding affinity of the proteins, bio-layer interferometry (BLI) measurements were performed on an Octet K2 system (Pall FortéBio, Fremont, CA, USA). Streptavidin-conjugated (SA) biosensors were soaked in Kinetic buffer (10 mM Na₂HPO₄, 10 mM NaH₂PO₄, 150 mM NaCl, 0.002% Tween20, 0.1% BSA, pH 7.4) for 10 min. After that, 100 nM biotinylated His₆ peptide (bio-EGGGSHHHHHH-COOH, synthesized as in Figure S1, Supplementary materials) was loaded on an SA biosensor and equilibrated in Kinetic buffer before analysis. For each measurement, 80 μL of unlabeled protein or Q-body in Kinetic buffer in two-fold gradient concentrations were applied, which was followed by the cycle of: 1000 rpm shake speed, baseline measurement in Kinetic buffer for 1 min, association measurement in a sample for 200~400 s, and dissociation measurement in Kinetic Buffer for 200~400 s. The biosensors were regenerated in 500 mM phosphoric acid (pH 1.0) for 1 min and equilibrated with Kinetic buffer for 30 s before the next cycle. The data were imported into Data Acquisition 11.0 (FortéBio) and the kinetic constants were calculated by Data Analysis 11.0 (FortéBio) using double reference subtraction assuming a 1:1 binding model.

The binding kinetics of the SOSIP constructs against a panel of IgG’s were determined via BLI on an Octet Red system (FortèBio). Anti-human IgG Fc capture biosensors were presoaked in binding buffer (phosphate-buffered saline (PBS pH 7.4) supplemented with 0.1% BSA, 0.005% Tween 20, and 0.02% NaN3) for 10 min. The hydrated tips were then loaded with purified IgG prepared at 8 μg/mL in binding buffer for 80 s. After reaching a stable baseline, antibody-immobilized biosensors were moved into wells containing a 2-fold dilution series of SOSIP trimer to monitor association for 3 min, then biosensors were moved back into wells containing binding buffer to monitor dissociation for 3 min. Responses were calculated and double referenced against the buffer reference signal and non-specific binding of analyte to biosensor in absence of IgG. Kinetic data were analyzed by using FortéBio Data Analysis 11.0 software and were processed by Savitzky-Golay filtering prior to fitting using a 1:1 binding model. Reported values are averages of data repeated in at least two independent experiments.

BLI binding experiments were performed using Octet RED96 equipment (ForteBio). The recombinant RBD (SinoBiological, 40592-V05H) and recombinant human ACE2 proteins (SinoBiological, 10108-H02H) were captured with an anti-mIgG Fc Capture (AMC) probe in PBST solution and then incubated with recombinant human ApoE2 (PeproTech, 350-12-500UG), ApoE3 (PeproTech, 350-02-500UG) and ApoE4 (PeproTech, 350-04-500UG) proteins. Then, the fully reacted solid-phase conjugates were dissociated in PBST buffer for analysis. The kinetic values were fitted to a 1:1 Langmuir binding model. The results were analysed with ForteBio Data Analysis 11.0 software to determine the association rate, dissociation rate and affinity constant.