Five genetically engineered mice that have a full set of human immunoglobulin variable region genes (Intelliselect transgenic mice)39 were immunized with PTx. This study was carried out under the Project Licenses 70/8718 issued by the UK Government Home Office under Animal (Scientific Procedures) Act (A(SP)A), 1986, incorporating Directive 2010/63/EU of the European Parliament, and with the approval of the Sanger Institute Animal Welfare and Ethical Review Body. The institute complied with the Code of Practice issued by the UK Government which aids compliance with the A(SP)A. The institute has a PHS assurance F16-00128 (WTSI).

A total of 1290 paired (VH/VL) sequences were recovered from antigen-sorted, plasma and memory cells via a previously published method.55 These 1290 sequences were expressed in HEK293 cells. Antibody supernatant was collected on d 8 after transfection and screened for binding to wildtype PTx by HTRF. Positive control anti-PTx antibody (ab37574, abcam) was diluted in Expi293TM Expression Medium (Gibco) over an 11-point titration using one in three dilutions to generate a standard curve. Titrations of 5 μl of antibody ab37574 were added to a 384-well white-walled assay plate (Greiner Bio-One). Negative control wells received 5 μl of Expi293TM Expression Medium only. Five icroliters of HEK293 antibody supernatants (undetermined concentration) were added to one well of the 384-well plate. Five microliters of PTx conjugated to Alexa 647 (Lightning-Link, Innova Bioscience) (3.75 nM final concentration) was added to all wells of the assay plate except negative control wells, which instead received 5 μl Expi293TM Expression Medium. Finally, 10 μl of anti-mouse IgG donor antibody (Southern Biotech; to bind to murine constant region in control and chimeric Intelliselect transgenic mouse antibodies) labeled with europium cryptate (Cis Bio), (1:4000 final concentration) was added to each well and the assay was left in the dark at room temperature to incubate for 2 h. After incubation, the assay was read on an Envision plate reader (Perkin Elmer) using a standard HTRF protocol. Then, 620 and 665 nm channel values were exported to Microsoft Excel (Microsoft) and F calculations performed ((665/620 nm ratio – signal negative control)/signal negative control) × 100). Percent effect values were calculated for each antibody by comparing its F value against a positive control antibody (ab37574) at 6.66 nM. The number of PTx-positive antibodies as a function of percentage effect values are shown in Figure 7a.

(a) Number of binders among the 1290 antibodies from the single-cell experiment as a function of percentage effect value relative to the positive control antibody, ab37574. At the chosen percentage effect of 10%, 364 of the 1290 binders are deemed PTx reactive. (b) Affinity measurements across the 364 PTx-reactive antibodies; antibodies in purple were used as probe antibodies for repertoire mining as per the “Repertoire mining experiment” subsection under the “Materials and methods” section

Antibody sequences with greater than 10% effect value relative to the positive control were labeled as binders. This resulted in 364 PTx-binders and 926 non-binders. SPR was performed on each of the 364 binders (Figure 7b).

Heavy chains from sorted splenic B cells from the same individual five mice as the single-cell data set were sequenced using standard protocols25 and processed using the pRESTO/Change-O pipeline.56,57 This resulted in 259,151 heavy chain sequences. For quality control, only sequences with read count (reads with a particular unique molecular identifier (UMI)) above or equal to two or a consensus count (reads with different UMIs but the same nucleotide sequence) above or equal to 10 were considered, reducing the size of the data set to 59,107 sequences.

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