A simple method for the evaluation of receptor binding capacity of modified cytokines.
We have developed a flow cytometric method to evaluate the binding of interleukin-2 analogues to receptors. The method relies on competition for binding between a fluorescein-conjugated monoclonal antibody (MoAb) directed against the human interleukin-2 receptor alpha chain (fluorescein isothiocyanate (FITC) anti-IL-2R) and the test protein. IL-2R positive cells are incubated with FITC-anti-IL-2R MoAb in the presence of native IL-2 or IL-2 iodinated by either the chloramine-T or the lactoperoxidase-glucose-oxidase method. The binding of IL-2 is indicated by decreased fluorescence. This method is suitable for measuring the binding capacity of modified IL-2 molecules and avoids the need for radioactive tracers. It provides a simple and reproducible technique, which can be extended readily to the study of the receptor binding capacity of cytokines conjugated with toxins, drugs or other molecules.
Chianelli
M
M
Clinica Medica II, University La Sapienza, Rome, Italy.
Signore
A
A
Hicks
R
R
Testi
R
R
Negri
M
M
Beverley
P C
PC
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001667:Binding, Competitive; D016207:Cytokines / Q000378:metabolism*; D005069:Evaluation Studies as Topic; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D005453:Fluorescence; D006801:Humans; D007376:Interleukin-2 / Q000378:metabolism; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D008722:Methods; D010835:Phytohemagglutinins / Q000494:pharmacology; D015375:Receptors, Interleukin-2 / Q000378:metabolism*; D013268:Stimulation, Chemical
D000911:Antibodies, Monoclonal; D016207:Cytokines; D007376:Interleukin-2; D010835:Phytohemagglutinins; D015375:Receptors, Interleukin-2; D016650:Fluorescein-5-isothiocyanate