Sequential determination of ligands binding to discrete components in heterogeneous mixtures by iterative panning and blocking (IPAB).

Biopanning has been used extensively in conjunction with purified components, but there are also examples in which mixtures of targets have been investigated. This study introduces a methodological innovation, termed iterative panning and blocking (IPAB), to extend the range of specific interactions that can be probed in mixtures. Here this procedure is used to probe a mixture of high molecular mass components of human cord blood with phage-peptide display libraries. The initial panning recovered phage that bore the consensus motif Gly-Pro-Arg-Pro, a known fibrinogen-binding motif. These phage bound specifically to purified fibrinogen. A series of peptides containing the Gly-Pro-Arg-Pro motif efficiently blocked the binding of phage having the same motif, presumably by binding to their common target. A second round of panning was performed against the same target mixture in the presence of this blocking peptide. Phage recovered from this second panning exhibited a motif (Ser-His-Tyr) that was subsequently shown to bind specifically to complement component C1q. A second peptide containing this motif specifically blocked the interaction of the phage with C1q. A third round of panning performed in the presence of both the fibrinogen- and the C1q- blocking peptides yielded phage with a new peptide motif (Asn-Pro-Phe) that also bound specifically to C1q, apparently at a new site. The three motifs isolated through this iterative process were distinct in that each was blocked only by its corresponding peptide. This IPAB strategy can be applied to many high diversity selection procedures that target complex mixtures.