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

På svenska

Author

  • Roope J. Huttunen
  • Tomas C. O'Riordan
  • Pirkko Härkönen
  • Juhani T. Soini
  • Niko J. Meltola
  • Pekka E. Hanninen
  • Aleksi E. Soini

Summary, in English

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 (TNF alpha). 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:13 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. Copyright (C) 2007 John Wiley & Sons, Ltd.

Publishing year

2007

Language

English

Pages

163-170

Publication/Series

Luminescence

Volume

22

Issue

3

Document type

Journal article

Publisher

John Wiley & Sons Inc.

Topic

  • Cancer and Oncology

Keywords

  • endothelial cells
  • ICAM-1
  • time-resolved fluorescence
  • immunocytochemistry
  • protein expression analysis
  • drug discovery

Status

Published

ISBN/ISSN/Other

  • ISSN: 1522-7243