human cd19 microbeads  (Miltenyi Biotec)

Figure S1 . " title="... shown in dot plots as percentages of total CD19 + cells. Left: IgG + CD27 + cells ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: SARS-CoV-2 spike-specific mAb binding profiles (A) Cells recovered from two sorting strategies, shown in dot plots as percentages of total CD19 + cells. Left: IgG + CD27 + cells from 18 donors and the subset of those that expressed S-binding BCRs. Right: cells from three donors expressing S-binding BCRs and sorted to recover principally those that did not bind RBD. (B) Summary of all productive mAbs (recombinant human IgG1) screened by ELISA (with recombinant S ectodomain trimer) and cell-surface expression assays (both 293T and yeast cells). Total numbers in the center of each of pie chart; numbers and color codes for the indicated populations shown next to each chart. To the right of the charts for the two sorting strategies are bar graphs showing frequencies of SARS-CoV-2 RBD and NTD binding mAbs for those subjects from whom at least ten paired-chain BCR sequences were recovered. (C) Binding to a panel of S proteins and SARS-CoV-2 subdomains, listed on the left, as determined by both ELISA and by association with S expressed on the surface of 293T cells or with RBD or NTD expressed on the surface of yeast cells, for S + sorted (left) and S + RBD − sorted cells (right). Left panel, 157 clones bound to S and an additional one bound to only RBD but not S. Pink indicates ELISA screens. Blue indicates cell-based screens. Each short section of a row represents an antibody. The rows labeled VH mutation and VL mutation are heatmaps of counts (excluding CDR3) from alignment by IgBLAST, with the scale indicated. (D) Dot plots of VH and VL mutation counts in mAbs that bound RBD, NTD, S2, and a “broad CoV group” that included MERS, HKU1, and OC43. ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; nonparametric Kruskal-Wallis multiple comparison. Horizontal lines show mean ± SEM. See also Figure S1 .

Techniques Used: Binding Assay, Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, Clone Assay, Labeling, Mutagenesis, Comparison

Figure 1 (A) Representative flow cytometry plots showing CD19 + , CD27 + , SARS-CoV-2 S-binding B cells from a convalescent subject (C12, top row) and a pre-pandemic control (bottom row). PBMCs were pre-enriched with CD19 magnetic beads then gated on live IgD − IgM-IgG + CD27 + and finally on S (B) Representative flow cytometry plots showing S-positive, RBD-negative B cells for three convalescent subjects and a pre-pandemic control, sorted as in (A) except for the S gate. (C) Representative flow plot of mAb supernatant bound to SARS-CoV-2 S on HEK293T cells. Cells were gated on DAPI − GFP + population. (D) Representative flow plot of mAb supernatant bound to SARS-CoV-2 RBD on yeast. cMyc tag indicated yeast that expressed RBD. (E) Representative flow plot of mAb supernatant bound to SARS-CoV-2 NTD on yeast. cMyc tag indicated yeast that expressed NTD. See Figure 1 C for the screening color scheme. (F) Bar graph of Log 10 (EC 50 ) of antibodies targeting RBD, NTD and S2 using ELISA and cell-based assay. EC 50 (μg/mL), RBD (n = 23), NTD clusters (n = 15) and S2 (n = 15). ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; Paired nonparametric Wilcoxon test. Data are mean values ± SEM (G) Dot plot of Log 10 (EC 50 ) of antibodies in the indicated bins by cell-based assay. Antibodies are from subjects G32 and C41, sorted with S. Each dot represents one monoclonal antibody. EC 50 (μg/mL), 13-39 days (n = 13), 40-63 days (n = 8). No significance; nonparametric Mann-Whitney test. Data are mean values ± SEM. " title="... rid="fig1">Figure 1 (A) Representative flow cytometry plots showing CD19 + , CD27 + , SARS-CoV-2 S-binding B ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: Sorting strategy for SARS-CoV-2-specific memory B cells and characterization of monoclonal antibodies, related to Figure 1 (A) Representative flow cytometry plots showing CD19 + , CD27 + , SARS-CoV-2 S-binding B cells from a convalescent subject (C12, top row) and a pre-pandemic control (bottom row). PBMCs were pre-enriched with CD19 magnetic beads then gated on live IgD − IgM-IgG + CD27 + and finally on S (B) Representative flow cytometry plots showing S-positive, RBD-negative B cells for three convalescent subjects and a pre-pandemic control, sorted as in (A) except for the S gate. (C) Representative flow plot of mAb supernatant bound to SARS-CoV-2 S on HEK293T cells. Cells were gated on DAPI − GFP + population. (D) Representative flow plot of mAb supernatant bound to SARS-CoV-2 RBD on yeast. cMyc tag indicated yeast that expressed RBD. (E) Representative flow plot of mAb supernatant bound to SARS-CoV-2 NTD on yeast. cMyc tag indicated yeast that expressed NTD. See Figure 1 C for the screening color scheme. (F) Bar graph of Log 10 (EC 50 ) of antibodies targeting RBD, NTD and S2 using ELISA and cell-based assay. EC 50 (μg/mL), RBD (n = 23), NTD clusters (n = 15) and S2 (n = 15). ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; Paired nonparametric Wilcoxon test. Data are mean values ± SEM (G) Dot plot of Log 10 (EC 50 ) of antibodies in the indicated bins by cell-based assay. Antibodies are from subjects G32 and C41, sorted with S. Each dot represents one monoclonal antibody. EC 50 (μg/mL), 13-39 days (n = 13), 40-63 days (n = 8). No significance; nonparametric Mann-Whitney test. Data are mean values ± SEM.

Techniques Used: Bioprocessing, Flow Cytometry, Binding Assay, Control, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Cell Based Assay, MANN-WHITNEY

Figure Legend Snippet:

Techniques Used: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Saline, Random Hexamer, Reverse Transcription, Transfection, Luciferase, Plasmid Preparation, Flow Cytometry, Software