When characterizing phage clones by the ELISA protocol in the manual, it is difficult to add more than 1012 virions per 100 µL well. This corresponds to a phage concentration of only 16 nM. At this concentration, an unambiguously positive ELISA signal can only be observed if the binding affinity is in the micromolar range or better. The iterative nature of phage selection permits identification of ligands with a broad range of affinities, from subnanomolar to 1 millimolar, so lower affinity ligands will not show a positive ELISA signal. In this case it is necessary to increase the concentration of the selected ligand, either by synthesing a peptide corresponding to the selected sequence (be sure to include the spacer sequence GGGS at the C-terminus, and amidate the C-terminal carboxylate if possible), or by expressing the selected sequence as an N-terminal fusion to a smaller protein. Alternatively, a sandwich ELISA can be carried out in which the selected phage is immobilized and an excess of target applied in the liquid phase. This procedure requires an antibody against the target protein, or some other means of detecting bound target protein. Coat the wells overnight with anti-M13 antibody (no HRP), wash, and add serial dilutions of each phage clone (one clone per row). After 1 hour, wash away unbound phage and add an excess of target protein (0.1 - 1 µM) in TBST. Incubate 1-2 hours at RT, wash away unbound target, and detect bound target with an enzyme-linked antibody.