Figure 1d:
Graph characterizations of NIR fluorescence HER2 imaging probe. (a) Saturation probe receptor assay for cyanine 5.5–trastuzumab binding to HER2/neu-overexpressing SKBR-3 cells. Inset illustrates Scatchard analysis of probe binding. Bmax = maximal bound probe concentration (in nanomolars), f(x) = linear relationship of the Scatchard plot. (b) Competitive inhibition of cyanine 5.5–trastuzumab binding with increasing molar concentrations of nonlabeled trastuzumab (Herceptin) or nonspecific human immunoglobulin G (Ig). (c) Probe-binding affinity as a function of fluorescent dye–to-protein ratio. (d) Representative flow cytometry histograms of cultured SKBR-3 cells without (open red-outlined spectrum) and with (filled red-outlined spectrum) addition of 2.5 nmol/L cyanine 5.5–conjugated HER2 probe. To demonstrate probe specificity, the histogram of 9L cells (green) treated with a fivefold higher (12.5 nmol/L) probe concentration is also shown. Max = maximum. (e) Flow cytometry summary of HER2 probe binding. Mean fluorescence intensity, with standard error of mean, for tumor cells without and with 100 ng of HER2 probe are shown. For competitive inhibition experiments, a 100-fold molar excess of unlabeled trastuzumab was added to the cells simultaneously with the probe. au = arbitrary units, ** = significant difference (P < .01).