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

Area of Science:

• Biochemistry
• Cell Biology
• Analytical Chemistry

Background:

• Accurate quantification of cell surface protein expression is crucial for understanding cellular functions and disease mechanisms.
• Existing methods may have limitations in sensitivity, throughput, or imaging capabilities.

Purpose of the Study:

• To introduce and validate a novel method for quantitative detection of cell surface protein expression.
• To assess the performance of this method using specific protein targets and compare it with conventional labeling techniques.

Main Methods:

• The method employs immunocytochemistry combined with time-resolved fluorimetry using long decay time europium(III) chelate and platinum(II) porphyrin labels.
• Adhered cells are measured in a dry state after assay completion, enabling repeated analysis and microscopy.
• Intercellular adhesion molecule-1 (ICAM-1) expression in EAhy926 endothelial cells, induced by tumor necrosis factor-alpha (TNFα), was used as a model.

Main Results:

• The novel method achieved superior signal-to-background (S:B) ratios of 20 for europium and 9 for platinum labels, compared to 3 for a standard FITC label.
• Excellent screening assay performance was indicated by Z'-factors greater than 0.5 for all tested labels.
• The protocol allows for postponed analysis and microscopy imaging of samples.

Conclusions:

• The developed method provides a robust and sensitive approach for quantitative protein expression analysis.
• Its suitability for both high-throughput screening and detailed fluorescent microscopy imaging makes it a versatile tool.