High-throughput SPR binding kinetics

The procedure for measuring CoVIC antibody construct binding kinetics was described previously (54). Briefly, the measurements were done on the Carterra LSA platform using HC30M sensor chips (Carterra) at 25°C, with a single analyte titrated against multiple CoVIC antibody constructs in each assay. Antibody constructs were either captured by amine-coupled goat anti-Human IgG Fc secondary antibody (Millipore) or directly amine-coupled to sensor chips depending on whether the constructs were monoclonal IgG antibodies or not. Antigens were injected in a twofold dilution series onto the chip surface from the lowest to the highest concentration without regeneration, preceded by several injections of buffer for signal stabilization. The highest concentration used for each antigen construct was as follows: RBD 40 µg/mL (1.11 µM), NTD 320 µg/mL (5.71 µM), D614-HexaPro 100 µg/mL (0.181 µM), D614G-HexaPro 100 µg/mL (0.170 µM), B.1.351-HexaPro 100 µg/mL (0.170 µM) ,and BA.1-HexaPro 200 µg/mL (0.351 µM). For each concentration, the cycle times included 120 seconds of baseline, 300 seconds of association, and 900 seconds of dissociation.

The titration data were pre-processed using the Kinetics (Carterra) software before exporting, and then analyzed using the TitrationAnalysis tool developed in-house (75). For each CoVIC antibody construct–antigen pair, the averaged k_a, k_d, and K_D values from 1:1 Langmuir model fitting were calculated for the best triplicate measurements satisfying the preset data acceptance criteria: (i) standard error of the estimated k_a, k_d, and K_D in each replicate was ≤20% and 2) the fold change for all three parameters within the triplicate was ≤3.