A Novel Affinity Tag, ABTAG, and Its Application to the Affinity Screening of Single-Domain Antibodies Selected by Phage Display
| Autor: | Toya Nath Baral, Shalini Raphael, Henk van Faassen, Kevin A. Henry, Greg Hussack, Jason Baardsnes, C. Roger MacKenzie, Jianbing Zhang |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
lcsh:Immunologic diseases. Allergy
0301 basic medicine Phage display Immunology VHH 03 medical and health sciences Microtiter plate 0302 clinical medicine Immunology and Allergy Bovine serum albumin Surface plasmon resonance Panning (camera) Original Research single-domain antibody biology Heavy-chain antibody antibody discovery Fusion protein Molecular biology carcinoembryonic antigen-related cell adhesion molecule 6 nanobody 030104 developmental biology Single-domain antibody 030220 oncology & carcinogenesis biology.protein phage display lcsh:RC581-607 surface plasmon resonance |
| Zdroj: | Frontiers in Immunology Frontiers in Immunology, Vol 8 (2017) |
| ISSN: | 1664-3224 |
| Popis: | ABTAG is a camelid single-domain antibody (sdAb) that binds to bovine serum albumin (BSA) with low picomolar affinity. In surface plasmon resonance (SPR) analyses using BSA surfaces, bound ABTAG can be completely dissociated from the BSA surfaces at low pH, over multiple cycles, without any reduction in the capacity of the BSA surfaces to bind ABTAG. A moderate throughput, SPR-based, antibody screening assay exploiting the unique features of ABTAG is described. Anti-carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) sdAbs were isolated from a phage-displayed sdAb library derived from the heavy chain antibody repertoire of a llama immunized with CEACAM6. Following one or two rounds of panning, enriched clones were expressed as ABTAG fusions in microtiter plate cultures. The sdAb-ABTAG fusions from culture supernatants were captured on BSA surfaces and CEACAM6 antigen was then bound to the captured molecules. The SPR screening method gives a read-out of relative expression levels of the fusion proteins and kinetic and affinity constants for CEACAM6 binding by the captured molecules. The library was also panned and screened by conventional methods and positive clones were subcloned and expressed for SPR analysis. Compared to conventional panning and screening, the SPR-based ABTAG method yielded a considerably higher diversity of binders, some with affinities that were three orders of magnitude higher affinity than those identified by conventional panning. |
| Databáze: | OpenAIRE |
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