A, Comparative blood incubation assay of radiolabeled preparations of F8-IL12p40 C175S and F8 diabody. Equimolar concentrations of the proteins were incubated in murine blood for 1 hour at 37°C. After centrifugation, radioactivity content of supernatant and pellet was measured. B, SDS-PAGE analysis of purified F8-IL12p40 C175S before (1) and after (2) deglycosylation using PNGase F. The deglycosylated product was purified over protein A (3) to separate it from the PNGase F enzyme (m: marker; r: reducing conditions; nr: non-reducing conditions). C, Size exclusion chromatogram. D, Mass spectrometric analysis (ESI/LC-MS). E, Wavelength scan and thermal denaturation curve assessed by CD spectroscopy. F, Quantitative biodistribution analysis F8-IL12p40 C175S in F9 teratocarcinoma bearing mice, 24 h after injection of 10 μg radioiodinated protein preparation. Radioactivity content of each organ is shown as percent injected dose per gram [%ID/g].