Dataset Information

Application of phage display to high throughput antibody generation and characterization.

ABSTRACT: We have created a high quality phage display library containing over 1010 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry assay. Furthermore, positive staining by immunohistochemistry on tissue microarrays was found for 37% (143/381) of antibodies. Thus, we have demonstrated the potential of and illuminated the issues associated with genome-wide monoclonal antibody generation.

SUBMITTER: Schofield DJ

PROVIDER: S-EPMC2258204 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Application of phage display to high throughput antibody generation and characterization.

Schofield Darren J DJ Pope Anthony R AR Clementel Veronica V Buckell Jenny J Chapple Susan Dj SDj Clarke Kay F KF Conquer Jennie S JS Crofts Anna M AM Crowther Sandra R E SR Dyson Michael R MR Flack Gillian G Griffin Gareth J GJ Hooks Yvette Y Howat William J WJ Kolb-Kokocinski Anja A Kunze Susan S Martin Cecile D CD Maslen Gareth L GL Mitchell Joanne N JN O'Sullivan Maureen M Perera Rajika L RL Roake Wendy W Shadbolt S Paul SP Vincent Karen J KJ Warford Anthony A Wilson Wendy E WE Xie Jane J Young Joyce L JL McCafferty John J

Genome biology 20070101 11

We have created a high quality phage display library containing over 1010 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry a ...[more]

PMID: 18047641

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Project description:BackgroundCombinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i) the counting of transducing units and (ii) the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges.Methodology/principal findingsWe gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU), with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at >1,000-fold faster rate, and reduce costs approximately 250-fold for generating 10(6) ligand sequences.Conclusions/significanceOur analyses demonstrates that whereas this approach correlates with the traditional colony-counting, it is also capable of a much larger sampling, allowing a faster, less expensive, more accurate and consistent analysis of phage enrichment. Overall, qPhage plus pyrosequencing is superior to TU-counting plus Sanger sequencing and is proposed as the method of choice over a broad range of phage display applications in vitro, in cells, and in vivo.

Dataset Information

Application of phage display to high throughput antibody generation and characterization.

Publications

Application of phage display to high throughput antibody generation and characterization.

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