Refined multivalent display of bacterial spore-binding peptides

A multiple antigen peptide display scaffold was used to create multivalent versions of a heptapeptide selected previously by phage display to bind to Bacillus subtilis spores. A simple flow cytometric assay was developed in which a biotinylated form of the peptide was first bound to fluorescent streptavidin, then the fluorescent streptavidin-peptide complex was bound to spores before introduction into the cytometer. This assay clearly demonstrated that the tetravalent scaffold enhanced the affinity for B. subtilis spores by greater than 1 and 2 orders of magnitude when compared to divalent and monovalent analogues, respectively. However, variations in the number and flexibility of spacer residues within the scaffold did not significantly affect the binding affinity of the tetravalent peptides. Similar to prior reports, these multivalent scaffolds are effective most likely because they mimic the multivalent display of the original peptide library on the phage coat. Moreover, the tetravalent peptides can be readily integrated into a variety of heterogeneous and homogeneous spore-detection assay formats.