A multiparameter screening assay to assess the cytokine-induced expression of endothelial cell adhesion molecules.
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 TNFalpha or IL-1beta. 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 assay-type 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.
Zerwes
Hans-Günter
HG
Novartis Pharma AG, CH-4002 Basel, Switzerland.
Peter
Jürg C
JC
Link
Marion
M
Gubler
Hanspeter
H
Scheel
Günther
G
D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D015815:Cell Adhesion Molecules / Q000096:biosynthesis* / Q000276:immunology; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology*; D019040:E-Selectin / Q000096:biosynthesis / Q000276:immunology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis / Q000276:immunology; D007375:Interleukin-1 / Q000494:pharmacology; D028581:Lanthanoid Series Elements / Q000737:chemistry; D016719:Radioimmunodetection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D019010:Vascular Cell Adhesion Molecule-1 / Q000096:biosynthesis / Q000276:immunology
D000906:Antibodies; D015815:Cell Adhesion Molecules; D016207:Cytokines; D019040:E-Selectin; D007375:Interleukin-1; D028581:Lanthanoid Series Elements; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1