Tandem phage-display for the identification of non-overlapping binding pairs of recombinant affinity reagents

The 'sandwich' binding format, which uses two reagents that can bind simultaneously to a given analyte, is the gold standard in diagnostics and many biochemical techniques. One of the bottlenecks in creating a sandwich assay is identifying pairs of reagents that bind non-competitively to t...

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Bibliographic Details
Published in:Nucleic acids research 2017-10, Vol.45 (18), p.e158-e158
Main Authors: Gorman, Kevin T, Roby, Lauren C, Giuffre, Allison, Huang, Renhua, Kay, Brian K
Format: Article
Language:English
Subjects:
Affinity Labels - metabolism
Cell Surface Display Techniques - methods
Enzyme-Linked Immunosorbent Assay
Genetic Techniques
High-Throughput Screening Assays - methods
Humans
Methods Online
Peptide Library
Polymerization
Protein Binding
Protein Interaction Domains and Motifs
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
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Summary:The 'sandwich' binding format, which uses two reagents that can bind simultaneously to a given analyte, is the gold standard in diagnostics and many biochemical techniques. One of the bottlenecks in creating a sandwich assay is identifying pairs of reagents that bind non-competitively to the target. To bridge this gap, we invented Megaprimer Shuffling for Tandem Affinity Reagents (MegaSTAR) to identify non-competitive binding pairs of recombinant affinity reagents through phage-display. The key innovation in MegaSTAR is the construction of a tandem library, in which two reagents are randomly-displayed on the phage surface. This is accomplished by using a pool of 300-nucleotide long 'megaprimers', which code for previously-selected reagents, to prime second strand synthesis of a single-stranded DNA template and generate millions of pair-wise combinations. The tandem library is then affinity selected to isolate pairs that both reagents contribute to binding the target. As a proof-of-concept, we used MegaSTAR to identify pairs of fibronectin type III monobodies for three human proteins. For each target, we could identify between five and fifteen unique pairs and successfully used a single pair in a sandwich assay. MegaSTAR is a versatile tool for generating sandwich ELISA-grade and bispecific reagents.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkx688