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LT, Perlee,; Christiansen, Jason; Dondero, R; Grimwade, Brian G.; Lejnine, Serguei; Mullenix, Michael C.; Shao, Weiping; Sorette, Martin; Tchernev, VT; Patel, Dhavalkumar D.; Kingsmore, SF

doi:10.1186/1477-5956-2-9

Cited by 52 publications

(14 citation statements)

References 15 publications

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“…In an effort to build upon our recent gene expression studies of peripheral blood cells [2], we used a novel high-throughput protein microarray platform [15,16] to measure the levels of 160 analytes in blood serum of SLE cases and controls. Two groups of SLE cases were studied.…”

Section: Resultsmentioning

confidence: 99%

“…The levels of 160 serum protein analytes were measured in serum aliquots (100 μl) from the cases and controls using custom dual-antibody sandwich immunoassay arrays, as described (Table S1) [15,16]. Briefly, monoclonal capture antibodies specific for each analyte were fixed to glass slides, with 12 replicate spots for each analyte.…”

Section: Methodsmentioning

confidence: 99%

“…Slides were then incubated with secondary biotinylated polyclonal antibodies, and signals were amplified using a “rolling circle” method [16]. Quality control measures included optimization of antibody pairs, the use of internal controls to minimize array-to-array variation, and standardized procedures of chip manufacturing [15,16]. Arrays were scanned using a Tecan LS200 (Tecan, Männedorf, Switzerland) and mean fluorescence intensities were generated with customized software.…”

Section: Methodsmentioning

confidence: 99%

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Elevated Serum Levels of Interferon-Regulated Chemokines Are Biomarkers for Active Human Systemic Lupus Erythematosus

et al. 2006

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BackgroundSystemic lupus erythematosus (SLE) is a serious systemic autoimmune disorder that affects multiple organ systems and is characterized by unpredictable flares of disease. Recent evidence indicates a role for type I interferon (IFN) in SLE pathogenesis; however, the downstream effects of IFN pathway activation are not well understood. Here we test the hypothesis that type I IFN-regulated proteins are present in the serum of SLE patients and correlate with disease activity.Methods and FindingsWe performed a comprehensive survey of the serologic proteome in human SLE and identified dysregulated levels of 30 cytokines, chemokines, growth factors, and soluble receptors. Particularly striking was the highly coordinated up-regulation of 12 inflammatory and/or homeostatic chemokines, molecules that direct the movement of leukocytes in the body. Most of the identified chemokines were inducible by type I IFN, and their levels correlated strongly with clinical and laboratory measures of disease activity.ConclusionsThese data suggest that severely disrupted chemokine gradients may contribute to the systemic autoimmunity observed in human SLE. Furthermore, the levels of serum chemokines may serve as convenient biomarkers for disease activity in lupus.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Elevated Serum Levels of Interferon-Regulated Chemokines Are Biomarkers for Active Human Systemic Lupus Erythematosus

et al. 2006

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“…Multiplexing is less important when only a handful of analytes are targeted or when cross reactivity is a problem. Cross reactivity can be a problem when multiplexing sandwich assays by pooling detection antibodies, as off-target binding by the detection antibodies is increasing likely with increasing analyte numbers (Perlee et al, 2004). Another approach which avoids the problem of cross reactivity is to use only one detection antibody per array, but reduce the volume of each array (Forrester et al, 2007) (Fig.…”

Section: Strategic Planningmentioning

confidence: 99%

Using Antibody Arrays to Measure Protein Abundance and Glycosylation: Considerations for Optimal Performance

2013

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Antibody arrays provide a valuable method of obtaining multiple protein measurements from low volumes of biological samples. Antibody arrays can be designed to target not only core protein abundances (relative or absolute abundances, depending on the availability of standards for calibration), but also protein post-translational modifications, provided antibodies or other affinity reagents are available to detect the modifications. Glycosylation is a common modification that has important and diverse functions, both in normal and disease biology. The methods for measuring glycan levels on multiple, specific proteins using antibody arrays and glycan-binding reagents have made significant progress. Here we describe practical approaches to develop, optimize and use antibody array assays to determine both protein abundances and glycosylation states. We cover the use of low-volume arrays to reduce sample consumption and a new way to improve the binding strength of particular glycan-binding reagents through multimerization. These methods could be useful for a wide range of biological studies in which glycosylation may be changing or affect protein function.

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“…Antibody microarray, an innovative proteomic technology, which can profile protein expression in a high-throughput way, represents a promising tool to such ends. Unlike 2-D PAGE, LC, and MS, which attempt to identify every protein within the sample, antibody proteomics is limited by the need for high-quality, specific antibodies of a reproducible affinity to be raised against the proteins of interest [2]. But in the case of cancer genes, CK (cytokine) or signal-transduction proteins, we can just study a smaller subset of them at much lower density.…”

Section: Introductionmentioning

confidence: 99%

Construction of an antibody microarray based on agarose‐coated slides

Liu

Zhang

et al. 2007

Electrophoresis

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Construction of an antibody microarray based on agarose-coated slidesThe antibody microarray, a high-throughput multiplex immunoassay method, has become a significant tool for quantitative proteomics studies. We describe here the strategies for optimizing the condition of antibody microarray building based on agarose-coated slides. In this study, modified glass slides were robotically printed with capture antibodies against monocyte chemoattractant protein 1 (MCP-1), then dilutions of the cytokine were applied to the arrays, and the protein was detected with biotin-labeled antibody coupled with Cy3-conjugated streptavidin. Thus a protein profiling microarray based on sandwich immunoassay has been established. Various factors in the production of antibody microarrays were analyzed: the capture antibody concentrations, shelf life of the postprinting slides, blocking buffers, and reproducibility of the system. A calibration curve with a correlation coefficient of 0.9995 was established which suggested that the matrix can retain arrayed proteins in near-quantitative fashion. The results revealed high signal uniformity and reproducibility with regard to intra-array (1.3%) and the interarray (8.7%) variation at the capture antibody concentration of 125 mg/mL. Besides, the printed arrays could be stored for at least two months without any apparent change of the performance parameters.

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Cited by 52 publications

References 15 publications

Elevated Serum Levels of Interferon-Regulated Chemokines Are Biomarkers for Active Human Systemic Lupus Erythematosus

Elevated Serum Levels of Interferon-Regulated Chemokines Are Biomarkers for Active Human Systemic Lupus Erythematosus

Using Antibody Arrays to Measure Protein Abundance and Glycosylation: Considerations for Optimal Performance

Construction of an antibody microarray based on agarose‐coated slides

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