Fig. 4: Identifying V_HHs for resynthesis.

a–d Method of selecting V_HHs for resynthesis. Plots show all V_HHs identified in selections containing the antigen OprM; each colored point is a distinct CDR3 clonotype. Points within yellow regions represent V_HHs likely to recognize the antigen of interest. Opaque points are V_HHs chosen for resynthesis. a Binary enrichment probability. For a V_HH significantly enriched in N antigen⁺ selections, indicates the probability of observing this V_HH to be significantly enriched in N antigen^– selections; see methods. b Percentile sum for antigen⁺ and antigen^– selections; see methods. c Geometric mean of the enrichment for antigen⁺ and antigen^– selections; see methods. d Geometric mean of the starting abundance and ending abundance within antigen⁺ selections. e, f Behavior of each V_HH candidate was examined across all selections; this inset shows CDR3 #7a08ae, which was tested in Fig. 5b below. e Trace of CDR3 abundance at each round of selection; each trace represents a different selection. Traces are colored according to whether the selection was OprM⁺ (orange), OprM^– (blue) or OprM status was unknown (gray). f Ending abundance vs. enrichment plot; each point is a distinct selection, colored using the same scheme as the left panel. g We selected 83 V_HHs targeting 5 distinct antigens (the flagellar filament “FliC”, the flagellar hook-basal body “FlgEHKL”, and the efflux pump-associated outer membrane porins OprM, OprN, and OprJ) for resynthesis. Of these, 55 were successfully expressed as fusions to human IgG1-Fc and tested for staining of P. aeruginosa by flow cytometry. Of these, 28 were validated by flow cytometry to have the predicted specificity. For panels (a–f), source data are provided as a Source Data file.