Optimization of Rolling‐Circle Amplified Protein Microarrays for Multiplexed Protein Profiling

Shao, Weiping; Zhou, Zhimin; Laroche, Isabelle; Liang, Hong; Zong, Qiuling; Patel, Dhavalkumar D.; Kingsmore, Stephen F.; Piccoli, Steven P.

doi:10.1155/s1110724303209268

Cited by 57 publications

(25 citation statements)

References 11 publications

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“…Miniaturization and parallelization of immunoassays address these problems, and protein microarrays using beads and planar surfaces that allow the sensitive detection of proteins from a low amount of sample material are available (2)(3)(4)23). Nevertheless, limitations caused by the cross-reactivity of antibodies and the availability of matched antibody pairs often make multiplexing of sandwich immunoassays difficult (24,25). Here we show that the use of paramagnetic beads for suspension bead array-based immunoassays opens the possibility to sequentially run multiplexed assays from the very same sample, and this approach allows circumventing incompatibility or cross-reactivity problems of antibodies.…”

Section: Biotin-pementioning

confidence: 99%

Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling

Poetz

Henzler

Hartmann

et al. 2010

Molecular & Cellular Proteomics

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Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibodycoated, magnetic suspension bead arrays. As a miniaturized immunoassay format, suspension bead array-based assays fulfill the criteria of the ambient analyte theory, and our experiments reveal that the analyte concentrations are not significantly changed. The value of sequential multiplex analyte capturing was demonstrated by probing tumor cell line lysates for the abundance of seven different receptor tyrosine kinases and their degree of phosphorylation and by measuring the complex phosphorylation pattern of the epidermal growth factor receptor in the same sample from the same cavity. Molecular & Cellular Proteomics 9:2474 -2481, 2010.Phosphorylation of proteins is an integral part of the signal transduction of eukaryotic cells as it modulates the activity of complex protein networks. Although Western blot-and immunoprecipitation-based MS approaches (1, 2) can lead to detailed insights into these processes, most of the integrated approaches only allow a static view of protein phosphorylation because they are not suitable for the screening of hundreds of samples. Either planar or bead array-based sandwich immunoassays can be used to analyze the quantity and activation state of signaling molecules in multiplex, enabling the systematic profiling of protein abundance and post-translational modifications (3-6) in hundreds of samples. However, multiplex immunoassays are only suitable for the simultaneous analysis of a limited number of proteins. The detection of comprehensive phosphorylation patterns is difficult as this involves assay systems that are incompatible with multiplexing.In principle, two sandwich immunoassay setups are possible for probing the phosphorylation state of a protein. The first setup applies a capture antibody specific for a non-modified part of the protein and uses a phosphorylation state-specific detection antibody. When applied to an array-based format, however, this setup does not allow for the simultaneous measurement of the abundance and the degree of phosphorylation (3, 4). A mixture of detection antibodies, one specific for the phosphorylation site and one specific for the nonmodified site of the protein, would bind simultaneously to the two different epitopes, and assay signals could not be further deconvoluted by the spatial or color code of the array. The second sandwich immunoassay setup for the analysis of protein phosphorylation applies a phosphorylation state-specific capture antibody and a protein-specific detection antibody. In such a setup, an anti-phosphotyrosine antibody (e.g. mAb 4G10) cannot be applied as a capture antibody because a huge variety of tyrosine phosphorylated proteins would be captured, and spe...

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Section: Biotin-pementioning

confidence: 99%

Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling

Poetz

Henzler

Hartmann

et al. 2010

Molecular & Cellular Proteomics

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“…Luminex assays are based on custom dualantibody sandwich immunoassay arrays using xMAP technology (multi-analyte profiling beads) enabling the detection and quantification of multiple protein targets simultaneously. 54,55 Of the 160 analytes measured, 30 showed significant differences between the 'IFN-high' SLE group and controls. The extent of expression level difference was bigger in 'IFN-high' SLE than in 'IFN-low SLE'; however, only 11 of 30 analytes were significantly different between 'IFN-high' and 'IFN-low' SLE.…”

Section: Ifn Signature In Serum Proteinmentioning

confidence: 99%

A compass that points to lupus: genetic studies on type I interferon pathway

Kyogoku

Tsuchiya

2007

Genes Immun

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It was more than 20 years ago that patients with systemic lupus erythematosus (SLE) were first reported to display elevated serum levels of type I interferon (IFN). Since then, extensive studies revealed a crucial role for type I IFN in SLE pathogenesis. The current model proposes that small increase of type I IFN production by plasmacytoid dendritic cells (pDCs) is sufficient to induce unabated activation of immature peripheral DCs. IFN-matured DCs select and activate autoreactive T cells and B cells, rather than deleting them, resulting in peripheral tolerance breakdown, a characteristic feature of SLE. Furthermore, immune complexes provide an amplification loop to pDCs for further IFN production. In the past 5 years, high-throughput technologies such as expression profiling and single-nucleotide polymorphism (SNP) typing established the role of altered type I IFN system in SLE, and a detailed picture of its molecular mechanisms is beginning to emerge. In this review, we discuss two major lines of genetics studies on type I IFN pathway related to human SLE: (1) expression profiling of IFN-responsive genes and (2) disease-associated SNPs of IFN-related genes, especially IRF5 (IFN-regulatory factor 5). Lastly, we discuss how such genetic alterations in type I IFN pathway fit in the current model of SLE pathogenesis.

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“…A presentation by Molecular Staging on high-throughput protein microarrays for disease sample analysis was given in the workshop. The platform and assay validation, along with some details of data analysis strategies, have been recently described (35)(36)(37). Selectivity, sensitivity, broad dynamic range, and reproducibility were the components emphasized in the method validation.…”

Section: Emerging Novel Assaysmentioning

confidence: 99%

Method Validation and Measurement of Biomarkers in Nonclinical and Clinical Samples in Drug Development: A Conference Report

Lee¹,

Weiner

Sailstad³

et al. 2005

Pharm Res

179

160

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Biomarkers are increasingly used in drug development to aid scientific and clinical decisions regarding the progress of candidate and marketed therapeutics. Biomarkers can improve the understanding of diseases as well as therapeutic and off-target effects of drugs. Early implementation of biomarker strategies thus promises to reduce costs and time-to-market as drugs proceed through increasingly costly and complex clinical development programs. The 2003 American Association of Pharmaceutical Sciences/Clinical Ligand Assay Society Biomarkers Workshop (Salt Lake City, UT, USA, October 24-25, 2003) addressed key issues in biomarker research, with an emphasis on the validation and implementation of biochemical biomarker assays, covering from preclinical discovery of efficacy and toxicity biomarkers through clinical and postmarketing implementation. This summary report of the workshop focuses on the major issues discussed during presentations and open forums and noted consensus achieved among the participants on topics from nomenclature to best practices. For example, it was agreed that because reliable and accurate data provide the basis for sound decision making, biomarker assays must be validated in a manner that enables the creation of such data. The nature of biomarker measurements often precludes direct application of regulatory guidelines established for clinical diagnostics or drug bioanalysis, and future guidance on biomarker assay validation should therefore be adaptable enough that validation criteria do not stifle creative biomarker solutions.

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Optimization of Rolling‐Circle Amplified Protein Microarrays for Multiplexed Protein Profiling

Cited by 57 publications

References 11 publications

Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling

Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling

A compass that points to lupus: genetic studies on type I interferon pathway

Method Validation and Measurement of Biomarkers in Nonclinical and Clinical Samples in Drug Development: A Conference Report

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