Screening of combinatorial peptide libraries for selection of peptide ligand useful in affinity purification of target proteins

Claim 1

In a method of selecting a ligand which will bind to a target substance, the ligand being in a population of individual random ligands bound to individual support materials, and the selection being with a labeled signal-providing detection system, the improvement which comprises incubating the population of random ligands with components from the detection system prior to any contact of the population of ligands with the target substance such that, after contact with the target substance, the reagents of the labeled detection system can then be contacted again with the ligand population and used to generate a different a different signal to allow detection of the ligand which binds to the target substance against the background of the random ligands which do not bind to the target substance.

Claim 18

A method of identifying peptide ligands which bind with a specific target protein comprising: (a) immobilizing randomized peptides on individual chromatographic supports to create a population of supports, each support bead having bound to its surface peptides substantially identical to each other; (b) contacting the supports of step (a) with a first antibody capable of specifically binding to the target protein, under conditions that allow both specific and nonspecific binding to the target protein, said first antibody also capable of binding specifically and non-specifically to both the supports and peptides on the support; (c) contacting the supports of step (b) with a second antibody capable of specifically binding to the first antibody and having conjugated thereto an enzyme capable of reacting with at least two different substrates to produce two different color changes, under conditions sufficient to bind the second antibody to the first antibody, said second antibody also capable of binding specifically and non-specifically to both the supports and peptides on the support; (d) adding a first substrate to the supports of step (c) under conditions sufficient to cause a first color change on individual supports having bound thereto the second antibody; (e) adding the target protein to the supports of step (d) under conditions sufficient to allow specific binding of the target protein to at least one of the peptides bound to the supports; (f) contacting the supports of step (e) with the first antibody of step (b) under conditions sufficient to bind that antibody specifically to the target protein; (g) adding the second antibody of step (c) having the enzyme conjugated thereto to the supports of step (f) under conditions sufficient to bind the second antibody specifically to the first antibody bound to the target protein bound to the peptides bound to the supports; (h) adding a second substrate to the supports of step (g) under conditions sufficient to cause a second and different color change which distinguishes those supports having bound thereto the peptides which bind with the target protein; (i) separating the supports having the peptide that binds to the target protein on the basis of the color change caused by the preceding step; and (j) sequencing the peptide on the support separated in step (j).