Extended Data Fig. 2: Agnostic-based approach for antibody isolation and selection, related to Fig. 2.

(A) Schematic workflow depicting the strategy used to isolate and select anti-Kp antibodies from human donors. Silhouette from https://publicdomainvectors.org/ (Creative Commons: CC0 1.0). (B) Gating strategy for single-cell sorting of total memory B cells. Flow cytometry plots show the sequential gating used to identify live cells, lymphocytes, singlets, CD19⁺B cells, CD19⁺CD27⁺IgD⁻ memory B cells, and CD19⁺CD27⁺IgD⁻IgM⁻ B cells (representing memory B cells expressing IgG, IgA, or IgE). Plots were analyzed using FlowJo v10 (BD Biosciences). (C) ELISA screening of secreted monoclonal antibodies for binding to Kp. Heatmap shows reactivity against two Kp ST147_NDM-1 clinical isolates (c.i.). Antibodies were considered positive when the OD₄₀₅ signal was at least 3-fold higher than the blank. (D) Gating strategy for the detection of mAb binding to live bacteria by flow cytometry. Plots illustrate sequential gating to identify bacterial cells, singlets, and Alexa Fluor 488⁺ stained bacteria. Plots were generated using NovoExpress v1.6.2 (Agilent Technologies).