Binding kinetics of soluble ligands to transmembrane proteins: comparing an optical biosensor and dynamic flow cytometry.
BACKGROUND
The kinetics of protein-protein interactions can be monitored with optical biosensors based on the principles of either surface plasmon resonance or mirror resonance. These methods are straightforward for soluble proteins, but not for proteins inserted in the plasma membrane.


METHODS
We monitored with an IASys biosensor system, based on a resonant mirror: (1) the binding of cells to an immobilized ligand, (2) the binding of a soluble ligand to immobilized cells, and (3) the binding of a soluble ligand to immobilized plasma membrane vesicles. For comparison, the kinetics of fluorescent antibody binding to intact cells were measured by dynamic flow cytometry.


RESULTS
With an optical biosensor, the useful configuration is the one based on immobilized plasma membrane vesicles. However, signals can be detected only for very abundant binding sites (>10(6) per cell). Dynamic flow cytometry allows the accurate determination of the k(on) and k(off) of antibody binding. The sensitivity of the method is two orders of magnitude better than with an optical biosensor.


CONCLUSIONS
Although biosensors constitute a method of choice for measuring the interactions between soluble proteins, they are not well suited for measuring the interaction between soluble proteins and membrane-embedded proteins. On the contrary, flow cytometry is well suited for such an application, when it is used in a dynamic mode.
Boulla
G
G
Laboratoire d'Immunologie Cellulaire, Paris, France.
Randriamampita
C
C
Raposo
G
G
Trautmann
A
A
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000915:Antibody Affinity; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000032:analysis; D001360:Avidin; D001402:B-Lymphocytes / Q000737:chemistry / Q000166:cytology; D015374:Biosensing Techniques / Q000379:methods*; D018801:CD2 Antigens / Q000032:analysis; D017252:CD3 Complex / Q000032:analysis; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D018914:Cells, Immobilized; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006801:Humans; D019169:Jurkat Cells / Q000737:chemistry / Q000166:cytology; D007700:Kinetics; D008024:Ligands; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism*; D009244:NAD+ Nucleosidase / Q000032:analysis; D011485:Protein Binding / Q000276:immunology; D012680:Sensitivity and Specificity; D012995:Solubility
D015703:Antigens, CD; D000943:Antigens, Differentiation; D018801:CD2 Antigens; D017252:CD3 Complex; D008024:Ligands; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D001360:Avidin; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1