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

Abstract:

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Gorman KT,Roby LC,Giuffre A,Huang R,Kay BK

doi

10.1093/nar/gkx688

subject

Has Abstract

pub_date

2017-10-13 00:00:00

pages

e158

issue

18

eissn

0305-1048

issn

1362-4962

pii

4056715

journal_volume

45

pub_type

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