Use of real-time polymerase chain reaction to identify cell- and tissue-type-selective peptides by phage display

Phage display approaches are used increasingly in efforts to identify cancer-specific binding peptides and antibodies. Phage-derived reagents are likely to have broad applications in diagnostic and research pathology. A critical element in the identification of cell or tissue-type-specific phage is the ability to reproducibly quantify bound or eluted phage at various stages of panning and screening procedures. Traditional biological assays of phage numbers such as plaque counting are commonly applied but are time-consuming, labor-intensive, and poorly reproducible. Moreover, enzyme immunoassays only support a subset of target types. Here, we report on the use of real-time polymerase chain reaction (PCR) (M13qPCR) in developing methods for identification of cell- and tissue-type-specific binding peptides. With M13qPCR, we demonstrate a >/=5 log(10) dynamic linear range with high reproducibility and significantly lower coefficients of variation (10 to 20%) than conventional methodology. Using M13qPCR in phage-panning experiments on live leukemia and prostate cancer cells, cancer-binding phage were identified. Similar results were obtained with conventional methodology such as flow cytometry. These results were extended to specific application of M13qPCR in panning phage libraries on tissue sections of prostate and breast cancer. With the PCR-based method, direct quantification of phage bound to tissue sections correlated well with staining intensity and yielded phage that bound to neoplastic and nonneoplastic epithelium. Thus, real-time PCR-based methodology significantly improves a number of aspects of conventional phage-panning protocols. Furthermore, identification of phage that bind specifically to diseased or cancerous tissue sections will likely be facilitated by this PCR-based approach.