MABF119 Sigma-AldrichAnti-EGFR Antibody, clone 528 (Azide-free)

The relation between the concentration of epidermal growth factor (EGF) receptor/kinase and effects of EGF on cell proliferation has been studied using variant A431 cells and antagonist anti-EGF receptor monoclonal antibodies. Clonal A431 cell variants selected for escape from the EGF-mediated growth inhibition of parental A431 cells all have reduced concentrations of EGF receptor/kinase; Harvey sarcoma virus-transformed A431 cells, which have escaped from EGF-mediated growth inhibition, also have reduced EGF receptors. Three clonal variants which have reacquired EGF-mediated growth inhibition have 2- to 4-fold more EGF receptor than their respective parent variant. A biphasic response with stimulation at low and inhibition at high concentrations of EGF was especially evident in revertants of clone 29. Three separate antagonist monoclonal anti-EGF receptor antibodies block the growth inhibitory effects of EGF and uncover EGF-mediated growth stimulation. These studies indicate that in A431 cell variants a continuum of ligand-activated EGF receptors determines proliferative responses from low concentrations of active receptors under basal conditions to intermediate concentrations causing growth stimulation to high concentrations, causing inhibition of cell proliferation.

Epidermal growth factor (EGF) at 3 nM maximally inhibits the proliferation of A431 epidermoid carcinoma cells. We show that at lower concentrations, in the range of 3-100 pM, EGF has a mitogenic effect on A431 cells. In the presence of 100 nM anti-EGF-receptor monoclonal IgG (designated 528), which inhibits A431 cell proliferation and blocks greater than 95% of EGF binding, EGF becomes mitogenic for A431 cells at concentrations up to 3 nM. These results suggest that a minor population of high-affinity EGF receptors may be involved in stimulation of A431 cell proliferation. Saturation binding assays with 125I-labeled EGF indicate that approximately equal to 0.1-0.2% of receptors for EGF are high-affinity receptors that bind EGF with an estimated Kd of 7 X 10(-11) M. This affinity is nearly 2 orders of magnitude higher than that of the remaining EGF receptors. Although A431 cell proliferation is maximally inhibited by nonsaturating amounts of EGF (3 nM), maximal inhibition by 528 IgG (approximately equal to 70% of maximal inhibition by EGF) requires saturating concentrations of antibody (approximately equal to 15 nM). Unlike EGF, rapid down-regulation is not observed with 528 IgG. These results indicate different mechanisms of growth inhibition of A431 cells by EGF and 528 IgG.