Figure 8.

Selection of antibody-expressing B cells in peripheral blood mononuclear cells (PBMC) samples from patients with melanoma. (A) Representative flow cytometric dot plots of cell sorting gating strategy to select melanoma patient B cells recognizing melanoma cell line surface antigen (Ag)-coated fluorescent beads. Viable (live/dead dye) human CD45+ PBMC incubated with antigen-coated beads were identified (left) and B cells (CD19/CD22+ -FITC) recognizing antigen-coated beads (APC) were selected for single cell sorting (right). (B) Agarose gels of Ig heavy (left) and light (right) chain DNA fragments amplified through nested PCR. Each gel lane represents PCR products derived from a single B cell. Fragments of the expected sizes (~650 bp for the heavy chain, ~500 bp for the light chain fragments) were extracted and sequenced. Red boxes indicate matching Ig heavy and light chain DNA fragments originating from the same single B cell. (C) Flow cytometric dot plots depicting binding of two single B cell-derived monoclonal antibody clones (MM_Ib, MM_IV) to melanoma cell antigen-coated fluorescent beads (SK-MEL-28 melanoma cells). The bead-selected clone (MM_IV) bound to antigen-coated beads while the non-specific one (MM_Ib) did not show reactivity. Background binding was detected with secondary antibody alone, while beads were recognized by the positive control anti-CSPG4 antibody.