Nannini, F;
Senicar, L;
Parekh, F;
Kong, KJ;
Kinna, A;
Bughda, R;
Sillibourne, J;
... Onuoha, SC; + view all
(2021)
Combining phage display with SMRTbell next-generation sequencing for the rapid discovery of functional scFv fragments.
MAbs
, 13
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, Article 1864084. 10.1080/19420862.2020.1864084.
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Abstract
Phage display technology in combination with next-generation sequencing (NGS) currently is a state-of-the-art method for the enrichment and isolation of monoclonal antibodies from diverse libraries. However, the current NGS methods employed for sequencing phage display libraries are limited by the short contiguous read lengths associated with second-generation sequencing platforms. Consequently, the identification of antibody sequences has conventionally been restricted to individual antibody domains or to the analysis of single domain binding moieties such as camelid VHH or cartilaginous fish IgNAR antibodies. In this study, we report the application of third-generation sequencing to address this limitation. We used single molecule real time (SMRT) sequencing coupled with hairpin adaptor loop ligation to facilitate the accurate interrogation of full-length single-chain Fv (scFv) libraries. Our method facilitated the rapid isolation and testing of scFv antibodies enriched from phage display libraries within days following panning. Two libraries against CD160 and CD123 were panned and monitored by NGS. Analysis of NGS antibody data sets led to the isolation of several functional scFv antibodies that were not identified by conventional panning and screening strategies. Our approach, which combines phage display selection of immune libraries with the full-length interrogation of scFv fragments, is an easy method to discover functional antibodies, with a range of affinities and biophysical characteristics.
| Type: | Article |
|---|---|
| Title: | Combining phage display with SMRTbell next-generation sequencing for the rapid discovery of functional scFv fragments |
| Location: | United States |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1080/19420862.2020.1864084 |
| Publisher version: | https://doi.org/10.1080/19420862.2020.1864084 |
| Language: | English |
| Additional information: | This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
| Keywords: | Antibody discovery, affinity modulation, long-read sequencing, phage display, rat immune libraries, scFv antibody |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute > Research Department of Haematology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10121531 |
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