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. 2025 Aug 12;16:1625813. doi: 10.3389/fimmu.2025.1625813

Figure 2.

A composite image showing multiple panels related to protein analysis. Panel A displays a protein gel with a lane labeled B7H6-His, showing protein bands at various molecular weights. Panel B features bar graphs illustrating the clonal forming unit counts for input and output across four panning rounds. Panel C presents two protein gels with multiple samples labeled with different codes like M53, M26, and others. Panel D shows a line graph of protein binding against monoclonal antibody concentration, with lines representing different samples and EC50 values listed. Panel E contains flow cytometry histograms comparing sample names and geometric means for A431 and A431(B7H6), accompanied by tables detailing each sample's geometric mean in APC-A.

Phage display-derived B7-H6-specific monoclonal antibodies show nanomolar affinity. (A) SDS-PAGE analysis of purified recombinant B7-H6-His protein under reducing conditions. (B) Phage display library screening. Input and output phage titers were quantified via bacterial colony counts. (C) Non-reducing SDS-PAGE of scFv-rFc monoclonal antibodies (2 μg/lane), confirming dimeric assembly. (D) ELISA-based affinity measurement. Plates coated with 5 μg/mL B7-H6-His were incubated with serially diluted antibodies (0.01–100 nM) and detected using HRP-conjugated goat anti-rabbit IgG (1:5,000). (E) Flow cytometric validation of antibody specificity. A431(B7-H6)+ (red line) and A431− (shaded histogram) cells were stained with 5 μg/mL B7-H6 mAbs and Cy5-conjugated goat anti-rabbit IgG (1:500).