Though the classic enzyme-linked immunosorbent assay (ELISA) offers a powerful analytical technique, the sandwich-format ELISA typically enhances the measurements of specificity and sensitivity to levels required for biomarker-based diagnostics. However, pairs of noncompetitive binders necessary to form the “sandwich” are typically identified through an inefficient “guess and check” process. To overcome this obstacle, we have developed a phage display protocol to directly select noncompetitive phage-displayed peptides for multiple clinically relevant targets. Briefly, phage displaying previously selected binding peptides are biotinylated and immobilized directly to a microtiter plate. After incubation with the target, a library of phage-displayed peptides is applied and washed to remove non-specific binding partners. Because the binding location of the known peptide is occupied, only phage capable of binding noncompetitively remain. The known phage is eluted off the plate and removed with streptavidin-coated magnetic beads, leaving selectants that bind the target at a different location from the first, converting a known binding partner into a sandwich binder. This method is rapid, inexpensive, and easily incorporated into conventional phage display processes. The technique has been used to discover peptide sandwich binders readily applicable to clinical assays and biosensors.

Direct selection of sandwich binding pairs with modified phage display technology

2019 Fall National Meeting

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