Extended Data Fig. 1: Multiplexed spike-binding detection assay.
From: Imprinting of serum neutralizing antibodies by Wuhan-1 mRNA vaccines
a, Gating strategy for the multiplexed spike-binding assay. After gating on singlets, beads pre-bound with the spike protein of different SARS-CoV-2 strains were separated based on their intensity of SPHERO™ yellow fluorophore. b, Representative flow cytometry plots of mAb CV3-25 binding to Wuhan-1, B.1.351, BA.1, and BA.5 spike-charged detection beads at the indicated antibody concentrations. c, A CV3-25 standard curve of binding to Wuhan-1, B.1.351, BA.1, and BA.5 spike-charged detection beads (pooled from two experiments, error bars denote standard deviations [SD]). d, Literature-based SARS-CoV-2 spike binding characteristics and escape mutations of the indicated mAbs to Wuhan-1, B.1.351, BA.1, and BA.5 spike proteins. CHK-265 (anti-CHIKV E2) was included as a negative control. +, positive binding; −, negative binding; amino acid substitutions indicate the mutations in the corresponding SARS-CoV-2 strain that results in loss of mAb binding. e, Representative flow cytometry plots of the indicated mAbs to Wuhan-1, B.1.351, BA.1, and BA.5 spike-charged detection beads (three experiments). f, Pearson’s correlation between the spike-binding and ELISA endpoint titres of the mAbs evaluated in d-e. R2 and P values are indicated. g, Antibody binding (top panels) and neutralizing activity (bottom panels) of naïve mouse sera and pre-pandemic human sera to Wuhan-1, BA.1, BA.5, and XBB.1.5 spike proteins and chimeric VSV pseudoviruses (n = 4, one experiment). Positive controls of serum collected from a mouse immunized with two doses of mRNA-1273 followed by two doses of mRNA-1273.529 (encodes BA.1 spike protein) and serum collected from a SARS-CoV-2 non-infected clinical trial participant immunized with three doses of mRNA-1273 and boosted with mRNA-1273.222 (Wuhan-1/BA.5 bivalent vaccine) are included (horizontal lines indicate mean values; dotted lines show the LOD; values at the LOD are plotted slightly below the LOD for visualization). −, negative SARS-CoV-2 exposure history; +, positive SARS-CoV-2 exposure history.