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.
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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 · 3.53 Impact Factor
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ABSTRACT: The determination of the protein composition is a challenging task, yet a valuable endeavor. Information that lies within the composition can help predicting pathogenic mechanisms and diseases. Therefore, a mandatory requirement is the accurate quantification. Protein microarrays are a promising alternative to complement mass spectrometry (MS) techniques. Nevertheless, effects in microarray production and assay development may lead to altered signal intensities. Techniques like NormaCurve provide a more robust quantification by taking the immobilized protein content into account. Printing dilution series can be used to construct a non-linear standard curve to estimate the amount of bound IgG, which helps to overrule the detection range of one order of magnitude. Standard protocols based on projects like the 'minimum information about a proteomics experiment' will help to improve quality and overall comparability.Current opinion in chemical biology 11/2013; 18C:16-20. DOI:10.1016/j.cbpa.2013.10.024 · 7.65 Impact Factor
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ABSTRACT: The brain is a vital organ and because it is well shielded from the outside environment, possibilities for non-invasive analysis are often limited. Instead, fluids taken from the spinal cord or circulatory system are preferred sources for the discovery of candidate markers within neurological diseases. In the context of multiple sclerosis (MS), we applied an affinity proteomic strategy and screened 22 plasma samples with 4,595 antibodies (3,450 genes) on bead arrays, then defined 375 antibodies (334 genes) for targeted analysis in a set of 172 samples, and finally used 101 antibodies (43 genes) on 443 plasma, as well as 573 cerebrospinal spinal fluid (CSF) samples. This revealed alteration of protein profiles in relation to MS subtypes for IRF8, IL7, METTL14, SLC30A7 and GAP43. Respective antibodies were subsequently used for immunofluorescence on human post-mortem brain tissue with MS pathology for expression and association analysis. There, antibodies for IRF8, IL7 and METTL14 stained neurons in proximity of lesions, which highlighted these candidate protein targets for further studies within MS and brain tissue. The affinity proteomic translation of profiles discovered by profiling human body fluids and tissue provides a powerful strategy to suggest additional candidates to studies of neurological disorders.Journal of Proteome Research 09/2014; 13(11). DOI:10.1021/pr500609e · 5.00 Impact Factor