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N Biotechnol. 2016 Sep 25;33(5 Pt A):574-81. doi: 10.1016/j.nbt.2015.12.003. Epub 2015 Dec 17.

Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality.

Author information

1
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.
2
Department of Biotechnology, Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany; YUMAB GmbH, Rebenring 33, 38106 Braunschweig, Germany.
3
Department of Biochemistry, Universität Kassel, Heinrich-Plett-Straße 40, 34132 Kassel, Germany.
4
Department of Biotechnology, Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany.
5
Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum (DKFZ) , Im Neuenheimer Feld 580, 69120 Heidelberg, Germany; Sciomics GmbH, Im Neuenheimer Feld 583, 69120 Heidelberg, Germany.
6
Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximilians-Universität München, Butenandtstr. 5, 81377 Munich, Germany.
7
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.
8
Department of Surgery, University Hospital Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg, Germany.
9
Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum (DKFZ) , Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. Electronic address: j.hoheisel@dkfz.de.

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.

PMID:
26709003
DOI:
10.1016/j.nbt.2015.12.003
[Indexed for MEDLINE]
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