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

✍ Scribed by Hans-Günter Zerwes; Jürg C. Peter; Marion Link; Hanspeter Gubler; Günther Scheel

• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 258 KB
• Volume: 304
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2002.5626

✦ Synopsis

Compounds which inhibit endothelial cell inflammatory responses are believed to be of therapeutic value. The cell adhesion molecules E-selectin, ICAM-1, and VCAM-1 play important roles in inflammatory reactions by mediating leukocyte-endothelial interactions. To identify compounds which inhibit the expression of these adhesion molecules following cytokine stimulation we developed an assay which measures E-selectin, ICAM-1, and VCAM-1 in the same experiment. For this, we have taken advantage of the technology of time-resolved fluorimetry, which allows detection of several parameters in parallel, employing anti-E-selectin antibody labeled with europium, and anti-ICAM-1 and anti-VCAM-1 labeled with samarium and terbium, respectively. These antibodies were used to detect the respective antigens in human endothelial cells stimulated with TNF␣ or IL-1␤. In cross-competition assays these antibodies were found to bind specifically to TNF-or IL-1-stimulated cells. This assay, in which three parameters are measured in the same experiment, proved to be robust with signal to noise ratios of 25-35 for E-Selectin, 4 -8 for ICAM-1, and 3-9 for VCAM-1. The assay proved to be reproducible in high-throughput screening. The experience with this assay demonstrates that multiple parameters can be measured in an enzyme-linked immunosorbent assaytype assay on cells by using time-resolved fluorimetry. The possibility of obtaining several parameters from one experiment is feasible under high-throughput screening conditions and is of interest for other experimental setups in which the simultaneous measurement of several parameters is desired.