title: Validation of affinity reagents using antigen microarrays.
abstract: There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.
authors: Science for Life Laboratory Stockholm, School of Biotechnology, KTH - Royal Institute of Technology, Box 1031, SE-171 21 Solna, Sweden.
authors: Ronald
authors: R
authors: Sjöberg
authors: Mårten
authors: M
authors: Sundberg
authors: Anna
authors: A
authors: Gundberg
authors: Asa
authors: A
authors: Sivertsson
authors: Jochen M
authors: JM
authors: Schwenk
authors: Mathias
authors: M
authors: Uhlén
authors: Peter
authors: P
authors: Nilsson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000941:Antigens / Q000276:immunology*; D001681:Biological Assay; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology*; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000276:immunology*; D018909:src Homology Domains
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D007128:Immunoglobulin Fragments; D007202:Indicators and Reagents; D011506:Proteins; D057127:Single-Chain Antibodies