title: Next-Generation Sequencing of a Single Domain Antibody Repertoire Reveals Quality of Phage Display Selected Candidates.
abstract: Next-Generation Sequencing and bioinformatics are powerful tools for analyzing the large number of DNA sequences present in an immune library. In this work, we constructed a cDNA library of single domain antibodies from a llama immunized with staphylococcal enterotoxin B. The resulting library was sequenced, resulting in approximately 8.5 million sequences with 5.4 million representing intact, useful sequences. The sequenced library was interrogated using sequences of known SEB-binding single domain antibodies from the library obtained through phage display panning methods in a previous study. New antibodies were identified, produced, and characterized, and were shown to have affinities and melting temperatures comparable to those obtained by traditional panning methods. This demonstrates the utility of using NGS as a complementary tool to phage-displayed biopanning as a means for rapidly obtaining additional antibodies from an immune library. It also shows that phage display, using a library of high diversity, is able to select high quality antibodies even when they are low in frequency.
authors.affiliation: American Society for Engineering Education, Postdoctoral Fellow at the US Naval Research Laboratory, Washington, DC, United States of America.
authors.forename: Kendrick B
authors.initials: KB
authors.lastname: Turner
authors.affiliation: American Society for Engineering Education, Science and Engineering Apprenticeship Participant at US Naval Research Laboratory, Washington, DC, United States of America.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Naciri
authors.affiliation: Center for Bio/molecular Science and Engineering, US Naval Research Laboratory, Washington, DC, United States of America.
authors.forename: Jinny L
authors.initials: JL
authors.lastname: Liu
authors.affiliation: Center for Bio/molecular Science and Engineering, US Naval Research Laboratory, Washington, DC, United States of America.
authors.forename: George P
authors.initials: GP
authors.lastname: Anderson
authors.affiliation: Center for Bio/molecular Science and Engineering, US Naval Research Laboratory, Washington, DC, United States of America.
authors.forename: Ellen R
authors.initials: ER
authors.lastname: Goldman
authors.affiliation: Center for Bio/molecular Science and Engineering, US Naval Research Laboratory, Washington, DC, United States of America.
authors.forename: Dan
authors.initials: D
authors.lastname: Zabetakis
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity; D061505:Cell Surface Display Techniques; D003001:Cloning, Molecular; D018628:Gene Dosage; D015870:Gene Expression; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D010802:Phylogeny; D011485:Protein Binding; D055550:Protein Stability; D016415:Sequence Alignment; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D019151:Peptide Library; D061905:Single-Domain Antibodies