title: Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality.
abstract: Many diagnostic and therapeutic concepts require antibodies of high specificity. Recombinant binder libraries and related selection approaches allow the efficient isolation of antibodies against almost every target of interest. Nevertheless, it cannot be guaranteed that selected antibodies perform well and interact specifically enough with analytes unless an elaborate characterisation is performed. Here, we present an approach to shorten this process by combining the selection of suitable antibodies with the identification of informative target molecules by means of antibody microarrays, thereby reducing the effort of antibody characterisation by concentrating on relevant molecules. In a pilot scheme, a library of 456 single-chain variable fragment (scFv) binders to 134 antigens was used. They were arranged in a microarray format and incubated with the protein content of clinical tissue samples isolated from pancreatic ductal adenocarcinoma and healthy pancreas, as well as recurrent and non-recurrent bladder tumours. We observed significant variation in the expression of the E3 ubiquitin-protein ligase (CHFR) as well as the glutamate receptor interacting protein 2 (GRIP2), for example, always with more than one of the scFvs binding to these targets. Only the relevant antibodies were then characterised further on antigen microarrays and by surface plasmon resonance experiments so as to select the most specific and highest affinity antibodies. These binders were in turn used to confirm a microarray result by immunohistochemistry analysis.
authors.affiliation: Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum (DKFZ) , Im Neuenheimer Feld 580, 69120 Heidelberg, Germany; Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximilians-Universität München, Butenandtstr. 5, 81377 Munich, Germany.
authors.forename: Janek
authors.initials: J
authors.lastname: Kibat
authors.affiliation: Department of Biotechnology, Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany; YUMAB GmbH, Rebenring 33, 38106 Braunschweig, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schirrmann
authors.affiliation: Department of Biochemistry, Universität Kassel, Heinrich-Plett-Straße 40, 34132 Kassel, Germany.
authors.forename: Matthias J
authors.initials: MJ
authors.lastname: Knape
authors.affiliation: Department of Biotechnology, Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany.
authors.forename: Saskia
authors.initials: S
authors.lastname: Helmsing
authors.affiliation: Department of Biotechnology, Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany.
authors.forename: Doris
authors.initials: D
authors.lastname: Meier
authors.affiliation: Department of Biotechnology, Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Hust
authors.affiliation: Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum (DKFZ) , Im Neuenheimer Feld 580, 69120 Heidelberg, Germany; Sciomics GmbH, Im Neuenheimer Feld 583, 69120 Heidelberg, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Schröder
authors.affiliation: Department of Biochemistry, Universität Kassel, Heinrich-Plett-Straße 40, 34132 Kassel, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Bertinetti
authors.affiliation: Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximilians-Universität München, Butenandtstr. 5, 81377 Munich, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Winter
authors.affiliation: The Novo Nordisk Foundation Centre for Protein Research, Protein Structure and Function Program, University of Copenhagen, Faculty of Health and Medical Sciences, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
authors.forename: Khalid
authors.initials: K
authors.lastname: Pardes
authors.affiliation: The Novo Nordisk Foundation Centre for Protein Research, Protein Structure and Function Program, University of Copenhagen, Faculty of Health and Medical Sciences, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
authors.forename: Mia
authors.initials: M
authors.lastname: Funk
authors.affiliation: The Novo Nordisk Foundation Centre for Protein Research, Protein Structure and Function Program, University of Copenhagen, Faculty of Health and Medical Sciences, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
authors.forename: Andrea
authors.initials: A
authors.lastname: Vala
authors.affiliation: Department of Surgery, University Hospital Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg, Germany.
authors.forename: Nathalia
authors.initials: N
authors.lastname: Giese
authors.affiliation: Department of Biochemistry, Universität Kassel, Heinrich-Plett-Straße 40, 34132 Kassel, Germany.
authors.forename: Friedrich W
authors.initials: FW
authors.lastname: Herberg
authors.affiliation: Department of Biotechnology, Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Dübel
authors.affiliation: Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum (DKFZ) , Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. Electronic address: j.hoheisel@dkfz.de.
authors.forename: Jörg D
authors.initials: JD
authors.lastname: Hoheisel
mesh_terms: D000918:Antibody Specificity; D001709:Biotechnology; D006801:Humans; D007150:Immunohistochemistry; D019151:Peptide Library; D010865:Pilot Projects; D040081:Protein Array Analysis; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000235:genetics
chemical_list: D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies