Cargando…

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 the target....

Descripción completa

Detalles Bibliográficos
Autores principales: Gorman, Kevin T., Roby, Lauren C., Giuffre, Allison, Huang, Renhua, Kay, Brian K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737338/
https://www.ncbi.nlm.nih.gov/pubmed/28985360
http://dx.doi.org/10.1093/nar/gkx688
Descripción
Sumario: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.