Research paperValidation of affinity reagents using antigen microarrays
Introduction
It is of great importance to have access to well-characterised and renewable affinity reagents within the different disciplines of life science. Even though there are several hundred thousand antibodies and other affinity reagents and binding molecules available, many lack a solid validation and this make them less useful for the research community 1, 2, 3. There are two principally different ways to validate affinity reagents, firstly using an application specific assay, such as Western blot, immunofluorescence-based confocal microscopy or immunohistochemistry, aimed to determine the functionality of an affinity reagent in a particular application. Secondly more generic binding assays, such as ELISA or surface plasmon resonance, aimed to determine the binding of the reagent to its antigen, usually performed using the antigen as ligand in the assay. For the latter application, microarrays provide an attractive possibility to perform multiplex assays with many antigens displayed on a single surface making it possible to determine the selectivity and specificity in a background of many related and unrelated antigens.
Recently, an international effort was initiated to generate renewable affinity reagents by various methods to SH2-containing human proteins 4, 5. There are 110 such proteins encoded in the human genome and there is a lack of reagents to many of these proteins. The effort took advantage of several different research groups developing different types of affinity reagents. Antigens corresponding to these proteins were produced either by the Structural Genomics Consortium (SGC) [6] or by the Human Protein Atlas (HPA) project 7, 8. As affinity reagents, antigen-purified polyclonal HPA antibodies were produced in rabbits, mouse monoclonal antibodies and recombinant single-chain variable fragment (scFv) antibodies were produced in five different academic laboratories.
We have previously used a microarray format with 14 identical subarrays with 384 antigens in each subarray, enabling the simultaneous analysis of 14 binders on each slide. This setup has for several years routinely been used to validate antibodies within the Human Protein Atlas project and more than 29,000 antibodies have been validated since 2005 with an approximate throughput of 150 new antibodies to validate every week. The challenge here has been to broaden the application by combining different antigens and different types of binders with different principles for detection and also in many cases with unknown concentrations.
Here, we have developed a microarray assay for validation of affinity reagents of different sources using a standardised assay involving a fluorescent read-out. The antigen microarray format developed here is a high-throughput and versatile validation tool for the analysis of the specificity of different affinity reagents being profiled with different antigens.
Section snippets
Antigen production
In total, 64 SH2-domain containing proteins were represented on the antigen microarrays. They were distributed among 85 different HPA antigens or as they are denoted Protein Epitope Signature Tags (PrESTs), which have been designed and expressed as protein fragments from the corresponding SH2-domain containing proteins and produced within the Human Protein Atlas project. These 85 PrESTs represent 53 unique proteins and were originally designed to have as low as possible similarity to other
Principle of the assay
There is a need to establish a platform for validation of affinity reagents from different sources and protein microarrays offer one such possibility. In Fig. 1, the main types of affinity reagents and antigens are schematically shown centred around the protein microarray platform. By immobilising relatively large numbers of different types of related and non-related antigens on the same array surface, a specificity profiling of antibodies and other binders is enabled. An antigen microarray as
Discussion
We have shown here the potential of antigen microarrays as an informative and important validation tool to analyse a wide variety of affinity reagents. To illustrate the feasibility and potential of the antigen microarray based platform, we have utilised binders from the international SH2-consortium formed to demonstrate the potential in systematic and high-throughput generation of antigens and corresponding affinity reagents 4, 5. This geographically well dispersed set of academic research
Acknowledgements
We are thankful for the collaborations with all participants within the SH2-consortium and especially here the providers of antigens and binding reagents: Dr. Susanne Gräslund et al., Structural Genomics Consortium; Dr. Alan Sawyer et al., Monash University; Professor Siqi Liu et al., Beijing Institute of Genomics; Professor Stefan Dübel et al., Technische Universität Braunschweig; Dr. John McCafferty et al., University of Cambridge.
This study was supported by AFFINOMICS, an EC FP7
References (18)
A genecentric Human Protein Atlas for expression profiles based on antibodies
Mol. Cell. Proteomics
(2008)Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3)
Protein Exp. Purif.
(2010)Towards proteome scale antibody selections using phage display
Nat. Biotechnol.
(2010)- et al.
Antibodypedia, a portal for sharing antibody and antigen validation data
Mol. Cell. Proteomics
(2008) Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics
Mol. Cell. Proteomics
(2007)ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome
Nat. Methods
(2007)Antibodies: the generation game
Nature
(2007)Antibody validation
Biotechniques
(2010)A roadmap to generate renewable protein binders to the human proteome
Nat. Methods
(2011)
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These authors contributed equally to this work.