Fig. 2: Isolation of anti-PstS1 mAbs from memory B cells of P.4.
a Donor P.4 serum responses taken at three different time points (indicated) to recombinant PstS1 by ELISA. b Isolation of PstS1-specific B cells. Whole-blood-derived lymphocytes were stained for CD19, membrane IgG, and PstS1. A total of 148 positive cells were single-cell sorted. c Pie charts representing the heavy chain sequences that were amplified from the CD19+/IgG+/PstS1+ B cells from P.4. The number in the middle of the pies denotes the total number of sequences, and the colored slices indicate clonally related sequences. Upper panel: all sequences. Lower panel: only the 16 clonally related sequences: clone 1—dark blue, clone 2—purple, clone 3—magenta, clone 4—teal, clone 5—green. d Nucleotide mutations in VH of the clonal versus nonclonal sequences. Error bars are represented as mean ± SEM. n = 16 clonal Vh sequences and n = 85 nonclonal Vh sequences. e, f are dendrograms (created using Geneious software) of the clonally related sequences, heavy and light chains, respectively. The mAbs selected for expression are colored in red. g–i The binding of nine mAbs by ELISA to recombinant PstS1 (g), Mtb-CDC1551 lysate (h), and Mtb-H37Rv lysate (i). j Comparing binding by ELISA of the mature (mt) and the predicted germline (gl) versions of p4-36 (magenta) and p4–163 (dark blue), to recombinant PstS1, Mtb-CDC1551 lysate, and Mtb-H37Rv lysate. AUC binding score of each antibody was determined as relative to the AUC of the mature antibody, which was normalized to 1 (see also Supplementary Fig. 5). All data are representative of at least two independent experiments (g–j).
