A Multiparameter Screening Assay to Assess the Cytokine-Induced Expression of Endothelial Cell Adhesion Molecules

Zerwes, Hans‐Günter; Peter, Jürg C.; Link, Marion; Gubler, Hanspeter; Scheel, Günther

doi:10.1006/abio.2002.5626

Cited by 16 publications

(11 citation statements)

References 12 publications

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“…Zerwes et al (5) recently described the use of Sm, Tb and Eu labels in a three-parameter expression analysis of plated HUVE cells. The authors applied immunocytochemistry with labelled primary antibodies and the DELFIA detection technique.…”

Section: Statistical Evaluation Of the Assay Performancementioning

confidence: 98%

“…More recently, upregulation of ICAM-1 by cytokines has raised increasing interest in the study of cell signalling (4). Also, upregulation of cell surface adhesion molecules has been used as a convenient means for the development of new methods for lead compound screening (5). In this study, we introduce a method for protein expression analysis.…”

Section: Detection Of Icam-1 On Endothelial Cellsmentioning

confidence: 99%

“…It has been demonstrated in the literature that luminescence signals from long decay time lanthanide (Sm, Tb and Eu) and metalloporphyrin (Pd and Pt porphyrins) labels can be efficiently discriminated from each other and also from conventional prompt fluorescent labels by their characteristic excitation and emission wavelengths, narrow luminescence bandwidths, and luminescence decay times (5,15,(20)(21)(22)(23). In cell expression analysis, the multiple colour detection strategy would be valuable not only in increasing the number of expression targets (number of parameters) but, more importantly, to reduce signal variability originating from variation in the cell plating density.…”

Section: Statistical Evaluation Of the Assay Performancementioning

confidence: 99%

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Quantitative detection of cell surface protein expression by time‐resolved fluorimetry

et al. 2007

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A method is introduced for quantitative detection of cell surface protein expression. The method is based on immunocytochemistry, the use of long decay time europium(III) chelate and platinum(II) porphyrin labels, and detection of photoluminescence emission from adhered cells by time-resolved fluorimetry. After immunocytochemistry, the assay wells are evaporated to dryness and measured in the dry state. This protocol allows repeated and postponed analysis and microscopy imaging. In order to investigate the performance of the method, we chose expression of intercellular adhesion molecule-1 (ICAM-1) of endothelial cell line EAhy926 as a research target. The expression of ICAM-1 on the cells was enhanced by introduction of a cytokine, tumour necrosis factor-alpha (TNFalpha). The method gave signal:background ratios (S:B) of 20 and 9 for europium and platinum labels, respectively, whereas prompt fluorescent FITC label gave a S:B of 3. Screening window coefficients (=Z'-factor) were >0.5 for all the three labels, thus indicating a score for an excellent screening assay. In conclusion, the method appears to be an appropriate choice for protein expression analysis, both in high-throughput screening applications, and for detailed sample investigation by fluorescent microscopy imaging.

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Section: Statistical Evaluation Of the Assay Performancementioning

confidence: 98%

Section: Detection Of Icam-1 On Endothelial Cellsmentioning

confidence: 99%

Section: Statistical Evaluation Of the Assay Performancementioning

confidence: 99%

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Quantitative detection of cell surface protein expression by time‐resolved fluorimetry

et al. 2007

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“…DELFIA technology is based on the time-resolved fluorescence of lanthanide chelates (15), and replacement of the colorimetric label employed by ELISA with lanthanide fluorescent labels utilized by DELFIA allows the detection of up to four labels simultaneously in a single well (10,17,18). Furthermore, the large linear range of the fluorescent signal requires testing of fewer sample dilutions, making the assay more suitable for a high-throughput format.…”

mentioning

confidence: 99%

Serological Responses to Experimental Norwalk Virus Infection Measured Using a Quantitative Duplex Time-Resolved Fluorescence Immunoassay

Kavanagh

Estes

Reeck

et al. 2011

Clin. Vaccine Immunol.

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A quantitative duplex time-resolved fluorescence assay, dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA), was developed to measure Norwalk virus (NV)-specific IgA and IgG antibodies simultaneously. The duplex assay showed superior performance by detecting seroconversion following experimental NV infection at an earlier time point than a reference total immunoglobulin enzyme-linked immunosorbent assay (ELISA).

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“…Despite the homeostatic benefits of endothelial cell activation in host defense, such activation also plays a central role in the pathogenesis of many chronic inflammatory disorders (32); this has resulted in chemical screens predominantly focused on the identification of compounds that decrease endothelial cell inflammation (33)(34)(35)(36)(37). In stark contrast, far less effort has been put forth to understand the underlying networks by which compounds may activate or inflame the endothelium.…”

mentioning

confidence: 99%

Diversity through phosphine catalysis identifies octahydro-1,6-naphthyridin-4-ones as activators of endothelium-driven immunity

Cruz

Wang²,

Kibbie

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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The endothelium plays a critical role in promoting inflammation in cardiovascular disease and other chronic inflammatory conditions, and many small-molecule screens have sought to identify agents that prevent endothelial cell activation. Conversely, an augmented immune response can be protective against microbial pathogens and in cancer immunotherapy. Yet, small-molecule screens to identify agents that induce endothelial cell activation have not been reported. In this regard, a bioassay was developed that identifies activated endothelium by its capacity to trigger macrophage inflammatory protein 1 beta from primary monocytes. Subsequently, a 642-compound library of 39 distinctive scaffolds generated by a diversity-oriented synthesis based on the nucleophilic phosphine catalysis was screened for small molecules that activated the endothelium. Among the active compounds identified, the major classes were synthesized through the sequence of phosphine-catalyzed annulation, Tebbe reaction, Diels–Alder reaction, and in some cases, hydrolysis. Ninety-six analogs of one particular class of compounds, octahydro-1,6-naphthyridin-4-ones, were efficiently prepared by a solid-phase split-and-pool technique and by solution phase analog synthesis. Structure-function analysis combined with transcriptional profiling of active and inactive octahydro-1,6-naphthyridin-4-one analogs identified inflammatory gene networks induced exclusively by the active compound. The identification of a family of chemical probes that augment innate immunity through endothelial cell activation provides a framework for understanding gene networks involved in endothelial inflammation as well as the development of novel endothelium-driven immunotherapeutic agents.

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A Multiparameter Screening Assay to Assess the Cytokine-Induced Expression of Endothelial Cell Adhesion Molecules

Cited by 16 publications

References 12 publications

Quantitative detection of cell surface protein expression by time‐resolved fluorimetry

Quantitative detection of cell surface protein expression by time‐resolved fluorimetry

Serological Responses to Experimental Norwalk Virus Infection Measured Using a Quantitative Duplex Time-Resolved Fluorescence Immunoassay

Diversity through phosphine catalysis identifies octahydro-1,6-naphthyridin-4-ones as activators of endothelium-driven immunity

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