Figure 7

Analysis of EGFR expression levels and binding affinities of sdAbs to human EGFR-presenting cells. Whole-cell lysates of exponentially growing cells (A431, FaDu, MDA-MB 435S) were prepared and equal amounts of total cellular proteins were separated by SDS-PAGE on 10% polyacrylamide gels. After Western Blot transfer onto PVDF membranes, EGFR and β-actin proteins were detected by incubation with the respective specific antibodies followed by HRP-coupled antibodies and chemiluminescence detection (A). In vitro specificity of ^99mTc-7C12 and ^99mTc-EG2 on A431 and FaDu cells was investigated after 1 h incubation on ice (B). Binding of radiolabeled sdAbs was blocked by 40-fold excess of unlabeled Cetuximab. Binding data is expressed as percent of injected dose per mg protein (% ID/mg protein). NB = non-blocked, B = blocked. For in vitro binding studies, two dimensional cultures of A431, FaDu and MDA-MB 435S cells were incubated with increasing concentrations of ^99mTc-7C12 (C). Total binding was measured in the absence of and nonspecific binding in the presence of 1 mM unlabeled sdAb. Specific binding was calculated as the difference between total and nonspecific binding. Binding studies were repeated twice and representative saturation curves for the EGFR-positive cell lines A431 and FaDu are shown. For the EGFR-negative cell line MDA-MB 435S, no specific binding was observed.