Characterization of an anti-CD20 camelid VHH and its expression on the Salmonella surface. (A) Fluorescence activated cell sorting dot plots of Namalwa cells stained with periplasmic lysates obtained from the indicated VHH-phage-producing E coli clones compared with a secondary/tertiary antibody negative control (Ab control) and an anti-CD20 monoclonal antibody positive control (CD20 mAb control). (B) Quantification of cell binding as determined via FACS using highly purified 2G9-6xHis-VHH or GSTb1-6xHis-VHH incubated with Namalwa CD20⁺ or 697 CD20⁻ cells. (C) Immunoprecipitation of native CD20 from intact cell membranes with either anti-CD20 rabbit polyclonal antibody (positive control), GSTb1-VHH negative control, or 2G9-VHH, as compared resin-only controls (Protein-A Sepharose or nickel nitrilotriacetic acid) with a lane containing the total protein before precipitation. (D) VHH expression on AT-Salmonella outer membrane fractions detected via the 6xHis tag (2G9 or GSTb1) or not (ompA empty vector) derived from equal cfus of bacterial pellets.