Screening Phage-Display Antibody Libraries Using Protein Arrays.

Phage-display technology constitutes a powerful tool for the generation of specific antibodies against a predefined antigen. The main advantages of phage-display technology in comparison to conventional hybridoma-based techniques are: (1) rapid generation time and (2) antibody selection against an unlimited number of molecules (biological or not). However, the main bottleneck with phage-display technology is the validation strategies employed to confirm the greatest number of antibody fragments. The development of new high-throughput (HT) techniques has helped overcome this great limitation. Here, we describe a new method based on an array technology that allows the deposition of hundreds to thousands of phages by micro-contact on a unique nitrocellulose surface. This setup comes in combination with bioinformatic approaches that enables simultaneous affinity screening in a HT format of antibody-displaying phages.

[1]  T. Waldmann,et al.  Immunotherapy: past, present and future , 2003, Nature Medicine.

[2]  A. Plückthun,et al.  Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. , 1988, Science.

[3]  Manqiu Cao,et al.  High-throughput generation and engineering of recombinant human antibodies. , 2001, Journal of immunological methods.

[4]  Emmanuel Dias-Neto,et al.  Next-Generation Phage Display: Integrating and Comparing Available Molecular Tools to Enable Cost-Effective High-Throughput Analysis , 2009, PloS one.

[5]  W. Huse,et al.  Discovery of human antibodies to cell surface antigens by capture lift screening of phage-expressed antibody libraries. , 1998, Analytical biochemistry.

[6]  Yanhui Hu,et al.  Next generation high density self assembling functional protein arrays , 2008, Nature Methods.

[7]  Johan T den Dunnen,et al.  Phage display screening without repetitious selection rounds. , 2012, Analytical biochemistry.

[8]  H. Lehrach,et al.  Protein microarrays for gene expression and antibody screening. , 1999, Analytical biochemistry.

[9]  M. Sheng,et al.  Antibodies in haystacks: how selection strategy influences the outcome of selection from molecular diversity libraries. , 2001, Journal of immunological methods.

[10]  M. Little,et al.  A surface expression vector for antibody screening. , 1991, Gene.

[11]  M. Murtaugh,et al.  High-level secretion of two antibody single chain Fv fragments by Pichia pastoris. , 1997, Journal of immunological methods.

[12]  H. Lehrach,et al.  3D protein microarrays: performing multiplex immunoassays on a single chip. , 2003, Analytical chemistry.

[13]  R. Kodzius,et al.  The powerful combination of phage surface display of cDNA libraries and high throughput screening. , 2012, Combinatorial chemistry & high throughput screening.

[14]  G. Winter,et al.  Improved tumour targeting by disulphide stabilized diabodies expressed in Pichia pastoris. , 1997, Protein engineering.

[15]  Noelia Dasilva,et al.  High-throughgput phage-display screening in array format. , 2015, Enzyme and microbial technology.

[16]  Michael Hust,et al.  Expression of Recombinant Antibodies , 2013, Front. Immunol..

[17]  G. Walter,et al.  Phage diabody repertoires for selection of large numbers of bispecific antibody fragments , 1996, Nature Biotechnology.

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

[19]  Lucy J. Holt,et al.  By-passing selection: direct screening for antibody-antigen interactions using protein arrays. , 2000, Nucleic acids research.

[20]  H. Hoogenboom,et al.  Selecting and screening recombinant antibody libraries , 2005, Nature Biotechnology.

[21]  J. Franconi,et al.  Identification of Human scFvs Targeting Atherosclerotic Lesions , 2006, Journal of Biological Chemistry.

[22]  Dieter Stoll,et al.  Protein microarrays for antibody profiling: Specificity and affinity determination on a chip , 2005, Proteomics.

[23]  H R Hoogenboom,et al.  Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. , 1991, Nucleic acids research.

[24]  A. Cattaneo,et al.  An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. , 1997, Gene.

[25]  D. Christ,et al.  Selection of human antibody fragments by phage display , 2007, Nature Protocols.

[26]  Jürgen Kreutzberger,et al.  Generation of High Density Protein Microarrays by Cell-free in Situ Expression of Unpurified PCR Products * , 2006, Molecular & Cellular Proteomics.

[27]  A. Wehnert,et al.  Rapid generation of functional human IgG antibodies derived from Fab-on-phage display libraries. , 2004, Journal of immunological methods.

[28]  Zoltán Konthur,et al.  Seeing better through a MIST: evaluation of monoclonal recombinant antibody fragments on microarrays. , 2004, Analytical chemistry.

[29]  L. Farinelli,et al.  By-passing in vitro screening—next generation sequencing technologies applied to antibody display and in silico candidate selection , 2010, Nucleic acids research.

[30]  Timo Pulli,et al.  Automated Panning and Screening Procedure on Microplates for Antibody Generation from Phage Display Libraries , 2009, Journal of biomolecular screening.

[31]  G. Murrell,et al.  Interferon-alpha (Intron A) upregulates urokinase-type plasminogen activator receptor gene expression , 2002, Cancer Immunology, Immunotherapy.

[32]  C. Milstein,et al.  Colony assays for antibody fragments expressed in bacteria. , 1991, Journal of immunological methods.

[33]  H. Lehrach,et al.  High-throughput screening of surface displayed gene products. , 2012, Combinatorial chemistry & high throughput screening.

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

[35]  Kendrick B. Turner,et al.  Next-Generation Sequencing of a Single Domain Antibody Repertoire Reveals Quality of Phage Display Selected Candidates , 2016, PloS one.

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

[37]  J. Hallborn,et al.  Automated screening procedure for high-throughput generation of antibody fragments. , 2002, BioTechniques.

[38]  N. Fischer,et al.  Sequencing antibody repertoires: the next generation. , 2011, mAbs.

[39]  G. Winter,et al.  Making antibodies by phage display technology. , 1994, Annual review of immunology.

[40]  Jan Berka,et al.  Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire , 2009, Proceedings of the National Academy of Sciences.

[41]  G. Winter,et al.  Filter screening of antibody Fab fragments secreted from individual bacterial colonies: specific detection of antigen binding with a two-membrane system. , 1991, Analytical biochemistry.

[42]  W. Huse,et al.  Cloning, isolation and characterization of human tumor in situ monoclonal antibodies , 2002, Cancer Immunology, Immunotherapy.

[43]  B. Kroesen,et al.  A rapid and versatile method for harnessing scFv antibody fragments with various biological effector functions. , 2000, Journal of immunological methods.

[44]  J. Hoheisel,et al.  Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality. , 2016, New biotechnology.

[45]  A. George,et al.  Antibody engineering: comparison of bacterial, yeast, insect and mammalian expression systems. , 1998, Journal of immunological methods.

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

[47]  M. Taussig,et al.  Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method). , 2001, Nucleic acids research.