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    Keywords: CANCER ; EXPRESSION ; CELL ; Germany ; GENOME ; microarray ; PROTEIN ; PROTEINS ; PATIENT ; COMPLEX ; COMPLEXES ; QUALITY ; BIOLOGY ; antibodies ; antibody ; ASSAY ; microarrays ; DESIGN ; ARRAYS ; REPRODUCIBILITY ; CANCER-PATIENTS ; CANCER PATIENTS ; pancreatic cancer ; PROTEOMICS ; PROTEOMIC ANALYSIS ; SERUM ; FEATURES ; PANCREATIC-CANCER ; antibody microarray ; HEALTHY-SUBJECTS ; methods ; HUMAN PLASMA ; DEPLETION ; QUANTITATIVE PROTEOMICS ; proteomic ; ABUNDANCE PROTEINS ; BIOMARKER DISCOVERY
    Abstract: Antibody microarrays have the potential to enable comprehensive proteomic analysis of small amounts of sample material. Here, protocols are presented for the production, quality assessment, and reproducible application of antibody microarrays in a two-color mode with an array of 1,800 features, representing 810 antibodies that were directed at 741 cancer-related proteins. In addition to measures of array quality, we implemented indicators for the accuracy and significance of dual-color detection. Dual-color measurements outperform a single-color approach concerning assay reproducibility and discriminative power. In the analysis of serum samples, depletion of high-abundance proteins did not improve technical assay quality. On the contrary, depletion introduced a strong bias in protein representation. In an initial study, we demonstrated the applicability of the protocols to proteins derived from urine samples. We identified differences between urine samples from pancreatic cancer patients and healthy subjects and between sexes. This study demonstrates that biomedically relevant data can be produced. As demonstrated by the thorough quality analysis, the dual-color antibody array approach proved to be competitive with other proteomic techniques and comparable in performance to transcriptional microarray analyses.
    Type of Publication: Journal article published
    PubMed ID: 20164060
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  • 3
    Keywords: CELLS ; BLOOD ; CELL ; evaluation ; Germany ; SYSTEM ; SYSTEMS ; GENOME ; microarray ; PROTEIN ; PROTEINS ; TIME ; COMPLEX ; COMPLEXES ; T cell ; T cells ; T-CELL ; T-CELLS ; BIOLOGY ; MOLECULAR-BIOLOGY ; LINKAGE ; SIGNAL ; cytokines ; antibodies ; antibody ; PERFORMANCE ; AMPLIFICATION ; MICROARRAY DATA ; microarrays ; DESIGN ; PLASMA ; CANCER-CELLS ; PARAMETERS ; Jun ; STRATEGIES ; sensitivity ; MICROARRAY ANALYSIS ; INTERFERON-GAMMA ; IMMUNOASSAYS ; PROTEOMICS ; CYTOKINE ; molecular biology ; molecular ; CHEMISTRY ; ELISA ; monitoring ; RHEUMATOID-ARTHRITIS ; antibody microarray ; CYTOKINE PRODUCTION ; LEVEL ; analysis ; methods ; ROLLING-CIRCLE AMPLIFICATION ; EXTENT ; SPECIMENS ; MATTER ; protein profiling ; microspot kinetics ; MASS-TRANSPORT ; quantitative ; detection strategies ; INTERLEUKIN-4 PRODUCTION ; PLASMA-PROTEOME ; TRITON X-100
    Abstract: Antibody microarrays have often had limited success in detection of low abundant proteins in complex specimens. Signal amplification systems improve this situation, but still are quite laborious and expensive. However, the issue of sensitivity is more likely a matter of kinetically appropriate microarray design as demonstrated previously. Hence, we re-examined in this study the suitability of simple and inexpensive detection approaches for highly sensitive antibody microarray analysis. N-hydroxysuccinimidyl ester (NHS)- and Universal Linkage System (ULS)-based fluorescein and biotin labels used as tags for subsequent detection with anti-fluorescein and extravidin, respectively, as well as fluorescent dyes were applied for analysis of blood plasma. Parameters modifying strongly the performance of microarray detection such as labeling conditions, incubation time, concentrations of anti-fluorescein and extravidin and extent of protein labeling were analyzed and optimized in this study. Indirect detection strategies whether based on NHS- or ULS-chemistries strongly outperformed direct fluorescent labeling and enabled detection of low abundant cytokines with many dozen-fold signal-to-noise ratios. Finally, particularly sensitive detection chemistry was applied to monitoring cytokine production of stimulated peripheral T cells. Microarray data were in accord with quantitative cytokine levels measured by ELISA and Luminex, demonstrating comparable reliability and femtomolar range sensitivity of the established microarray approach
    Type of Publication: Journal article published
    PubMed ID: 17474144
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  • 4
    Keywords: BLOOD ; Germany ; MODEL ; GENERATION ; SYSTEM ; SITE ; GENOME ; microarray ; TIME ; DNA ; MECHANISM ; cytokines ; antibodies ; antibody ; TRANSPORT ; ASSAY ; DESIGN ; PLASMA ; PARAMETERS ; KINETICS ; sensitivity ; RECEPTORS ; IMMUNOASSAYS ; immunoassay ; GUIDELINES ; LIGAND-BINDING ; CORRELATION SPECTROSCOPY ; CYTOKINE ; DEPENDENCE ; ANTIBODY MICROARRAYS ; SIZE ; TECHNOLOGY ; RELEVANCE ; GEOMETRY ; two-compartment model ; DNA HYBRIDIZATION ; mass transport ; microspot kinetics ; ANTIGEN-BINDING ; MASS-TRANSPORT ; PROTEIN ADSORPTION ; SOLID-SURFACES ; SURFACE-DIFFUSION
    Abstract: In this report we examine the limitations of existing microarray immunoassays and investigate how best to optimize them using theoretical and experimental approaches. Derived from DNA technology, microarray immunoassays present a major technological challenge with much greater physicochemical complexity. A key physicochemical limitation of the current generation of microarray immunoassays is a strong dependence of antibody microspot kinetics on the mass flux to the spot as was reported by us previously. In this report we analyze, theoretically and experimentally, the effects of microarray design parameters ( incubation vessel geometry, incubation time, stirring, spot size, antibody-binding site density, etc.) on microspot reaction kinetics and sensitivity. Using a two-compartment model, the quantitative descriptors of the microspot reaction were determined for different incubation and microarray design conditions. This analysis revealed profound mass transport limitations in the observed kinetics, which may be slowed down as much as hundreds of times compared with the solution kinetics. The data obtained were considered with relevance to microspot assay diffusional and adsorptive processes, enabling us to validate some of the underlying principles of the antibody microspot reaction mechanism and provide guidelines for optimal microspot immunoassay design. For an assay optimized to maximize the reaction velocity on a spot, we demonstrate sensitivities in the aM and low fM ranges for a system containing a representative sample of antigen-antibody pairs. In addition, a separate panel of low abundance cytokines in blood plasma was detected with remarkably high signal-to-noise ratios
    Type of Publication: Journal article published
    PubMed ID: 16735300
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  • 5
    Keywords: CELLS ; Germany ; HYBRIDIZATION ; microarray ; ANTIGEN ; SIMULATION ; BINDING ; antibodies ; TRANSPORT ; ASSAY ; microarrays ; KINETICS ; sensitivity ; IMMUNOASSAYS ; LINKED-IMMUNOSORBENT-ASSAY ; PROTEIN MICROARRAYS ; DIFFUSION ; antibody microarray ; ASSAYS ; ANTIBODY MICROARRAYS ; SOLID SUPPORTS ; adsorption ; two-compartment model ; mass transport ; BIOSENSORS ; mass-transport limited reaction ; microspot kinetics
    Abstract: It is well documented that diffusion has generally a strong effect on the binding kinetics in the microtiter plate immunoassays. However, a systematic quantitative experimental evaluation of the microspot kinetics is still missing in the literature. Our work aims at filling this important gap of knowledge on the example of antigen binding to antibody microspots. A mathematical model was derived within the framework of two-compartment model and applied to the quantitative analysis of the experimental data obtained for typical antibody microspot assays. A strong mass-transport dependence of the antigen-antibody microspot kinetics was identified to be one of the main restrictions of this new technology. The binding reactions are slowed down in the microspot immunoassays by several orders of magnitude as compared with the corresponding well-stirred bulk reactions. The task to relax the mass-transport limitations should thus be one of the most important issues in designing the antibody microarrays. These limitations notwithstanding, the detection range of more than five orders of magnitude and the high sensitivity in the low femtomolar range were experimentally achieved in our study, demonstrating thus an enormous potential of this highly capable technology
    Type of Publication: Journal article published
    PubMed ID: 16385475
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  • 6
    Keywords: APOPTOSIS ; CANCER ; CELL ; human ; KINASE ; SITES ; DISTINCT ; PROTEIN ; transcription ; DOMAIN ; SUFFICIENT ; nuclear bodies ; cell cycle ; CELL-CYCLE ; CYCLE ; fibroblasts ; PHOSPHORYLATION ; protein kinase ; PROTEIN-KINASE ; ISOFORM ; leukemia ; p53 ; RECRUITMENT ; CANCER-RESEARCH ; BODY ; serine ; DOMAINS ; ARCHITECTURE ; HIPK2 ; NUCLEAR-BODIES ; PML ; ACUTE PROMYELOCYTIC LEUKEMIA ; RAR-ALPHA ; SUMO-1
    Abstract: Here we demonstrate that endogenous human homeodomain-interacting protein kinase (HIPK) 2 and the highly homologous kinase HIPK3 are found in a novel sulmuclear domain, the HIPK domains. These are distinct from other sulmuclear structures such as Cajal bodies and nucleoli and show only a partial colocalization with promyelocytic leukemia (PML) nuclear bodies (PML-NBs). A kinase inactive HIPK2 point mutant is localized in the nucleoplasm. The occurrence of HIPK domains in PML-/- fibroblasts reveals their independence from the PML protein. HIPK2 can be almost completely recruited to PML-NBs by the PML isoform PML IV, but not by PML-III. PML IV-mediated recruitment of HIPK2 does not rely on its kinase function and also occurs in PML-/- fibroblasts, showing that this PML isoform is sufficient for recruitment of HIPK2. Whereas the architecture of HIPK domains is PML independent, HIPK2-mediated enhancement of p53-dependent transcription, p53 serine 46 phosphorylation and the antiprotiferative function of HIPK2 strictly rely on the presence of PML
    Type of Publication: Journal article published
    PubMed ID: 12907596
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