Unraveling the Properties of Phage Display Fab Libraries and Their Use in the Selection of Gliadin-Specific Probes by Applying High-Throughput Nanopore Sequencing

García Calvo, Eduardo Rafael; García García, Aina; Rodríguez Gómez, Santiago; Martín De Santos, María Del Rosario; García Lacarra, Teresa

Unraveling the Properties of Phage Display Fab Libraries and Their Use in the Selection of Gliadin-Specific Probes by Applying High-Throughput Nanopore Sequencing

Download

viruses-16-00686.pdf (4.19 MB)

Official URL

https://doi.org/10.3390/v16050686

Publication date

2024

Authors

García Calvo, Eduardo Rafael

García García, Aina

Rodríguez Gómez, Santiago

Martín De Santos, María Del Rosario

García Lacarra, Teresa

Publisher

MDPI

Citations

Exportar

URI

https://hdl.handle.net/20.500.14352/104803

Citation

Garcia-Calvo, E.; García-García, A.; Rodríguez, S.; Martín, R.; García, T. Unraveling the Properties of Phage Display Fab Libraries and Their Use in the Selection of Gliadin-Specific Probes by Applying High-Throughput Nanopore Sequencing. Viruses 2024, 16, 686. https://doi.org/10.3390/ v16050686

Abstract

Directed evolution is a pivotal strategy for new antibody discovery, which allowed the generation of high-affinity Fabs against gliadin from two antibody libraries in our previous studies. One of the libraries was exclusively derived from celiac patients’ mRNA (immune library) while the other was obtained through a protein engineering approach (semi-immune library). Recent advances in high-throughput DNA sequencing techniques are revolutionizing research across genomics, epigenomics, and transcriptomics. In the present work, an Oxford Nanopore in-lab sequencing device was used to comprehensively characterize the composition of the constructed libraries, both at the beginning and throughout the phage-mediated selection processes against gliadin. A customized analysis pipeline was used to select high-quality reads, annotate chain distribution, perform sequence analysis, and conduct statistical comparisons between the different selection rounds. Some immunological attributes of the most representative phage variants after the selection process were also determined. Sequencing results revealed the successful transfer of the celiac immune response features to the immune library and the antibodies derived from it, suggesting the crucial role of these features in guiding the selection of high-affinity recombinant Fabs against gliadin. In summary, high-throughput DNA sequencing has improved our understanding of the selection processes aimed at generating molecular binders against gliadin.