title: Next generation sequencing of all variable loops of synthetic single framework scFv-Application in anti-HDL antibody selections.
abstract: Next generation sequencing (NGS) can be applied to monitoring antibody phage display library selection processes to follow the enrichment of each individual antibody clone. Utilising the recent development of the Illumina sequencing platform enabling sequencing up to 2×300bp, we have developed a method to deep sequence all complementarity determining regions (CDRs) in the clones obtained from a synthetic single framework antibody library. This was complemented by an in-house bioinformatics pipeline for efficient analysis of the sequencing results. The method was utilised to study antibody selections against high density lipoprotein (HDL) particles. Sequencing of the output from each selection round enabled extraction of useful information on both the total copy numbers as well as the relative enrichment rates of the clones. Ten antibody clones showing different ranking in terms of frequency were reproduced from synthetic DNA constructs and their capacity to bind HDL was verified by an immunoassay. The method thus facilitates the isolation of clones of interest, and in particular can assist retrieval of less efficiently enriched, yet interesting clones, which are unlikely to be identified by conventional, random colony picking based, screening.
authors.affiliation: Department of Biochemistry, Division of Biotechnology, University of Turku, Turku, Finland. Electronic address: janita.lovgren@utu.fi.
authors.forename: Janita
authors.initials: J
authors.lastname: Lövgren
authors.affiliation: Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland.
authors.forename: Juha-Pekka
authors.initials: JP
authors.lastname: Pursiheimo
authors.affiliation: Department of Biochemistry, Division of Biotechnology, University of Turku, Turku, Finland.
authors.forename: Mikko
authors.initials: M
authors.lastname: Pyykkö
authors.affiliation: Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland.
authors.forename: Jussi
authors.initials: J
authors.lastname: Salmi
authors.affiliation: Department of Biochemistry, Division of Biotechnology, University of Turku, Turku, Finland.
authors.forename: Urpo
authors.initials: U
authors.lastname: Lamminmäki
mesh_terms: D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001483:Base Sequence; D001709:Biotechnology; D022801:Complementarity Determining Regions / Q000235:genetics / Q000276:immunology; D003327:Coronary Disease / Q000097:blood / Q000276:immunology; D005813:Genes, Synthetic; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007118:Immunoassay; D008075:Lipoproteins, HDL / Q000097:blood / Q000276:immunology*; D019151:Peptide Library; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology
chemical_list: D022801:Complementarity Determining Regions; D008075:Lipoproteins, HDL; D019151:Peptide Library; D057127:Single-Chain Antibodies
keywords: Antibody library; Deep sequencing; High density lipoprotein; Next generation sequencing; Phage display; scFv