Scintillation proximity assay to study the interaction of epidermal growth factor with its receptor.
Scintillation Proximity Assay (SPA), which does not require the physical separation of receptor bound and free ligand, was applied to study the interaction of Epidermal Growth Factor (EGF) with its receptor (EGFR) in membrane preparations from human placenta. Fluomicrospheres to which the monoclonal anti-EGFR antibody R1 was coupled, were used. Kinetic binding data of the association of 125I-labeled EGF binding to the receptor at 20 degrees C could be fitted according to a double exponential model, which is consistent with the presence of fast and slow associating EGF binding sites. Dissociation kinetics revealed that perturbation of equilibrium conditions rapidly occurs upon washing. Multiple point Scatchard analysis of equilibrium 125I-labeled EGF binding data revealed curvilinearity, indicating the presence of both high and low affinity EGF binding sites. We conclude that SPA is an interesting new tool in the exploration of the interaction of ligands with their receptors, which allows detailed ligand-receptor studies under precise in situ conditions.
Kienhuis
C B
CB
Department of Experimental and Chemical Endocrinology, University Hospital St., Radboud Nijmegen, The Netherlands.
Geurts-Moespot
A
A
Ross
H A
HA
Foekens
J A
JA
Swinkels
L M
LM
Koenders
P G
PG
Ireson
J C
JC
Benraad
T J
TJ
D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D005260:Female; D006801:Humans; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D008863:Microspheres; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011869:Radioligand Assay; D012588:Scintillation Counting / Q000379:methods*
D004815:Epidermal Growth Factor; D066246:ErbB Receptors