Ligand-receptor binding measured by laser-scanning imaging.
This report describes the integration of laser-scanning fluorometric cytometry and nonseparation ligand-binding techniques to provide new assay methods adaptable to miniaturization and high-throughput screening. Receptor-bound, cyanine dye-labeled ligands, [Cy]ligands, were discriminated from those free in solution by measuring the accumulated fluorescence associated with a receptor-containing particle. To illustrate the various binding formats accommodated by this technique, saturation- and competition-binding analyses were performed with [Cy]ligands and their cognate receptors expressed in CHO cells or as fusion proteins coated on polystyrene microspheres. We have successfully applied this technique to the analysis of G protein-coupled receptors, cytokine receptors, and SH2 domains. Multiparameter readouts from ligands labeled separately with Cy5 and Cy5.5 demonstrate the simultaneous analysis of two target receptors in a single well. In addition, laser-scanning cytometry has been used to assay enzymes such as phosphatases and in the development of single-step fluorescent immunoassays.
Zuck
P
P
Pharmacopeia Inc., CN5350, Princeton, NJ 08543, USA.
Lao
Z
Z
Skwish
S
S
Glickman
J F
JF
Yang
K
K
Burbaum
J
J
Inglese
J
J
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005434:Flow Cytometry; D051380:GRB2 Adaptor Protein; D019204:GTP-Binding Proteins / Q000032:analysis*; D007834:Lasers; D008024:Ligands; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000032:analysis*; D018121:Receptors, Cytokine / Q000032:analysis*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; D008024:Ligands; D011506:Proteins; D011956:Receptors, Cell Surface; D018121:Receptors, Cytokine; D010744:Phosphoric Monoester Hydrolases; D019204:GTP-Binding Proteins