Validation of affinity reagents using antigen microarrays

Science for Life Laboratory Stockholm, School of Biotechnology, KTH - Royal Institute of Technology, Box 1031, SE-171 21 Solna, Sweden.
New Biotechnology (Impact Factor: 2.9). 11/2011; 29(5):555-63. DOI: 10.1016/j.nbt.2011.11.009
Source: PubMed


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.

Download full-text


Available from: Peter Nilsson, Oct 31, 2014
25 Reads
  • Source
    • "(Tables 1 and 2). These microarrays have also proven to be useful tools for large scale screening of autoantibody targets in autoimmune diseases such as multiple sclerosis, as well as for generating binding profiles for different types of affinity binders (Fig. 1) [15] [16]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: High-density protein microarrays of recombinant human protein fragments, representing 12,412 unique Ensembl Gene IDs, have here been produced and explored. These protein microarrays were used to analyse antibody off-target interactions, as well as for profiling the human autoantibody repertoire in plasma against the antigens represented by the protein fragments. Affinity-purified polyclonal antibodies produced within the Human Protein Atlas (HPA) were analysed on microarrays of three different sizes, ranging from 384 antigens to 21,120 antigens, for evaluation of the antibody validation criteria in the HPA. Plasma samples from secondary progressive multiple sclerosis patients were also screened in order to explore the feasibility of these arrays for broad-scale profiling of autoantibody reactivity. Furthermore, analysis on these near proteome-wide microarrays was complemented with analysis on HuProt™ Human Proteome protein microarrays. The HPA recombinant protein microarray with 21,120 antigens and the HuProt™ Human Proteome protein microarray are currently the largest protein microarray platforms available to date. The results on these arrays show that the Human Protein Atlas antibodies have few off-target interactions if the antibody validation criteria are kept stringent and demonstrate that the HPA-produced high-density recombinant protein fragment microarrays allow for a high-throughput analysis of plasma for identification of possible autoantibody targets in the context of various autoimmune conditions.
    New Biotechnology 09/2015; DOI:10.1016/j.nbt.2015.09.002 · 2.90 Impact Factor
  • Source
    • "The presented study is an exploratory approach using antibody suspension bead arrays on a collection of serum samples from WD-SI-NET patients at different stages of disease. All antibodies used were routinely validated for specificity using planar protein microarrays against 384 protein antigens [14] as well as other methods [15]. The analytical format used here is a highly multiplexed single-binder immunoassay to enrich a protein in a complex solution, which yet cannot exclude off-target binding events. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Patients with well-differentiated small intestine neuroendocrine tumors (WD-SI-NETs) are most often diagnosed at a metastatic stage of disease, which reduces possibilities for a curative treatment. Thus new approaches for earlier detection and improved monitoring of the disease are required. Suspension bead arrays targeting 124 unique proteins with antibodies from the Human Protein Atlas were used to profile biotinylated serum samples. Discoveries from a cohort of 77 individuals were followed up in a cohort of 132 individuals both including healthy controls as well as patients with untreated primary WD-SI-NETs, lymph node metastases and liver metastases. A set of 20 antibodies suggested promising proteins for further verification based on technically verified statistical significance. Proceeding, we assessed the classification performance in an independent cohort of patient serum, achieving, classification accuracy of up to 85% with different subsets of antibodies in respective pairwise group comparisons. The protein profiles of nine targets, namely IGFBP2, IGF1, SHKBP1, ETS1, IL1α, STX2, MAML3, EGR3 and XIAP were verified as significant contributors to tumor classification. We propose new potential protein biomarker candidates for classifying WD-SI-NETs at different stage of disease. Further evaluation of these proteins in larger sample sets and with alternative approaches is needed in order to further improve our understanding of their functional relation to WD-SI-NETs and their eventual use in diagnostics.
    PLoS ONE 11/2013; 8(11):e81712. DOI:10.1371/journal.pone.0081712 · 3.23 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Affinity proteomics is the field of proteome analysis based on the use of antibodies and other binding reagents as protein-specific detection probes. In this review, the particular strengths of affinity methods for determination of protein localization, functional characterization, biomarker discovery and intracellular applications, and their resulting impact in basic and clinical research are highlighted. An additional focus is on the requirements for systematic binder generation and current large-scale binder projects, including bioinformatic frameworks for epitope selection and for documentation of available binding reagents and their performance. In addition to current affinity proteomics methods and applications, including arrays of proteins, binders, lysates and tissues, approaches coupling mass spectrometry-based proteomics and affinity proteomics are reviewed.
    Expert Review of Proteomics 08/2012; 9(4):401-14. DOI:10.1586/epr.12.34 · 2.90 Impact Factor
Show more