Figure 2. Selection of specific phage-displayed peptide ligands binding to myeloma Ig.

A: Enrichment of selectively binding phage on MM021 paraprotein over three selection rounds. A X₁₈ random peptide phage display library was screened on immobilized MM021 paraprotein. Bound phage were recovered by K91 bacterial infection, amplified overnight, purified and subjected to the next selection round. Negative preselection of the library was performed in selection round two and three. The selected phage were tested for binding to MM021 paraprotein and the control human polyclonal IgG after each selection round. Bacteria transduced by recovered phage were grown on LB plates containing tetracycline to determine the number of transducing units (TU) by colony counting. Ig = immunoglobulin, MM = Multiple Myeloma. B: Phage selected on myeloma Ig specifically bind the antibody on which they were selected. Examplarily, binding of paraprotein MM021-selected phage FLNGCDKEDWMCWVTT and control phage fd tet to MM021 monoclonal antibody, MM021 paraprotein and control human IgG is shown. Bound phage were quantified by Enzyme-linked Immunosorbent Assay (ELISA). Data are shown as means from triplicate experiments (± SEM). C: The protein GST-FLNGCDKEDWMCWVTT blocks binding of phage FLNGCDKEDWMCWVTT to MM021 paraprotein. MM021-selected phage FLNGCDKEDWMCWVTT was incubated on MM021 paraprotein in the presence of increasing amounts of GST-FLNGCDKEDWMCWVTT or GST alone. Bound phage were quantified as in B. Data are shown as relative values compared to binding of a control phage (means from triplicate experiments ± SEM). GST = glutathione S-transferase. D: Phage selected on myeloma Ig specifically bind their target and do not cross-react with other Igs. Binding of phage clones selected on the myeloma Ig of patients MM021, MM034 and MM036 as well as random control phage YMTPPLSSQQKS and fd tet to different myeloma paraproteins and recombinantly expressed Fab fragments (MM001, MM003, MM008, MM020) as well as control polyclonal IgG and Bovine Serum Albumin is shown. Bound phage were quantified as numbers of transducing units (TU) based on bacterial infection (means from triplicate platings ± SEM).