Detection of fibronectin expression by human endothelial cells using a enzyme-linked immunosorbent assay (ELISA): enzymatic degradation by activated plasminogen.
An enzyme-linked immunosorbent assay (ELISA) has been developed to measure cellular fibronectin (cFN) in association with human umbilical vein endothelial cells (HUVEC) in culture. The expression of a number of functional domains on the cFN molecule was demonstrated using three specific murine monoclonal antibodies. This system was found to be sensitive, detecting as little as 0.156 microg/ml of cFN, and required only 1.3 x 10(5) cells per well confluent cells per experimental condition. This allowed multiple experiments to be performed on one batch of endothelial cells. cFN was detected on both viable and methanol fixed endothelial cells without significant non-specific antibody binding. The utility of this experimental model was studied by exploring the effect of urokinase activated plasminogen, a potent protease, on the expression of cFN and its functional domains.
Daramola
O A
OA
Department of Paediatrics, Imperial College School of Medicine, London, UK.
Heyderman
R S
RS
Klein
N J
NJ
Shennan
G I
GI
Levin
M
M
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000276:immunology / Q000378:metabolism*; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D010958:Plasminogen / Q000494:pharmacology*; D013194:Staining and Labeling; D014471:Umbilical Veins
D000911:Antibodies, Monoclonal; D005353:Fibronectins; D010958:Plasminogen