High-throughgput phage-display screening in array format.

Emerging technologies for the design and generation of human antibodies require improved approaches enabling their screening, characterization and validation. Currently, strategies based on ELISA or western blot are used to that aim. However, the ever increasing number of novel antibodies generated would benefit from the development of new high-throughput (HT) platforms facilitating rapid antibody identification and characterization. Herein, we describe a protein chip bearing recombinant phage particles and based on a large phage antibody library. In this paper we have set forth a novel implementation which provides a powerful and simple methodology enabling the identification of single-chain variable fragments (scFv). As a proof-of-principle of this method, we tested it with recombinant antigen (human recombinant interleukin 8). Additionally, we developed a novel bioinformatics tool that serves to compare this novel strategy with traditional methods. The method described here, together with associated informatics tools, is robust, relatively fast and represents a step-forward in protocols including phage library screenings.

[1]  R. Fuller,et al.  Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences. , 2011, Cold Spring Harbor protocols.

[2]  D R Burton,et al.  Antibodies without immunization. , 1992, Science.

[3]  Manuel Fuentes,et al.  Nanotechniques in proteomics: protein microarrays and novel detection platforms. , 2012, European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences.

[4]  I. Tomlinson,et al.  Antibody arrays for high-throughput screening of antibody–antigen interactions , 2000, Nature Biotechnology.

[5]  B. Strukelj,et al.  Peptide Phage Display as a Tool for Drug Discovery: Targeting Membrane Receptors , 2011, Molecules.

[6]  Manuel Fuentes,et al.  Data Analysis Strategies for Protein Microarrays , 2012, Microarrays.

[7]  Mahavir Singh,et al.  Novel human recombinant antibodies against Mycobacterium tuberculosis antigen 85B , 2014, BMC Biotechnology.

[8]  G. Winter,et al.  Phage antibodies: filamentous phage displaying antibody variable domains , 1990, Nature.

[9]  Y. Ivarsson,et al.  Interaction Analysis through Proteomic Phage Display , 2014, BioMed research international.

[10]  G. P. Smith,et al.  Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. , 1985, Science.

[11]  M. Little,et al.  Human antibody libraries in Escherichia coli. , 1995, Journal of biotechnology.

[12]  J. C. Love,et al.  A microengraving method for rapid selection of single cells producing antigen-specific antibodies , 2006, Nature Biotechnology.

[13]  Noelia Dasilva,et al.  Biomarker Discovery by Novel Sensors Based on Nanoproteomics Approaches , 2012, Sensors.

[14]  Manuel Fuentes,et al.  New technologies in cancer. Protein microarrays for biomarker discovery , 2011, Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico.

[15]  Johan Ingvarsson,et al.  Microarrays based on affinity‐tagged single‐chain Fv antibodies: Sensitive detection of analyte in complex proteomes , 2005, Proteomics.

[16]  M. Trau,et al.  Duplex Microfluidic SERS Detection of Pathogen Antigens with Nanoyeast Single-Chain Variable Fragments , 2014, Analytical chemistry.