Preparation of biomolecules used as imaging agents for tumor detection.

<p>(A) Schematic drawing of antibody and antibody fragments (Fab), and serum albumin and streptavidin, illustrating size and shape of molecules. Approximate molecular weights are noted. (B-D) Coomassie blue stained 12% SDS-PAGE of protein A purified R6.5 IgG under non-reducing (NR) and reducing (R) conditions (B), digestion of R6.5 with papain, protein A elution, protein A flow products under reducing condition (C), all six biomolecules used in this study under non-reducing condition (D). The bands corresponding to the heavy and light chains of IgG (HC, LC) and, Fc, and Fab were labeled. (E) Flow cytometry histograms using constitutive ICAM-1 expressing HeLa cells and ICAM-1 negative HEK 293 cells labeled with Alexa488-labeled R6.5 (histograms in red line) and control IgG (histogram in blue line). Autofluorescence of cells without labeling is shown in solid filled histograms. (F) The level of binding (mean fluorescence intensity (MFI)) to HeLa cells of Alexa488-labeled R6.5 IgG and Fab was plotted against the concentration to estimate binding affinity. The solid line is the curve-fit of the Langmuir binding isotherm equation to data.