Wallac wizard

An aliquot of [¹⁸F]1 (0.37 MBq) was added to a mixture of PBS (pH 7.4) and n-octanol (2 mL each, n = 3) in polypropylene tubes. The mixture was vortexed for one minute, and the organic phase was separated by centrifugation (3000 rpm, 5 min). Next, aliquots of octanol (50 µL) and PBS (500 µL) were collected from each tube into preweighed Eppendorf^® tubes and measured for radioactivity in an automated gamma counter (Wallac Wizard™, Perkin Elmer, Waltham, MA, USA). Samples were weighed, and the weights were converted to volume according to solvent density. The partition coefficient value was calculated as the ratio of radioactivity (counts per minute (CPM)/mL) in octanol to that in the aqueous phase.

Cell uptake studies of [¹⁸F]1 were conducted using MCF-7 and HepG2 cell lines. Briefly, cells plated in 24-well plates (200,000/well) were treated with varying concentrations of SP-141 (1, 5, or 10 µM) or vehicle for 21 h and incubated with a fresh medium containing [¹⁸F]1 (~18.5 kBq in 0.5 mL medium) for 1 h. Next, the incubation medium was removed, cells were rinsed with cold PBS (3 × 0.5 mL) and lysed using Cell Culture Lysis Reagent (0.25 mL, Promega Corporation, Madison, WI, USA) followed by a rinse with PBS (0.125 mL/well). For each well, the supernatant, PBS washes, and the cell lysate were measured for radioactivity in an automated gamma counter (Wallac Wizard™, Perkin Elmer, Waltham, MA, USA). Subsequently, cell lysates were analyzed for protein concentration using the Bradford assay (Bio-Rad, Hercules, CA, USA). Cell uptake for each well was normalized to its protein content and expressed as % uptake/mg protein. Results are presented as mean ± SD for three wells.

Human serum was purchased (Biochrom, Biochrom, UK). Human plasma and cerebrospinal fluid was a kind gift from the laboratory of Prof. P. Lewczuk of the clinical neurochemistry in Erlangen. Rat plasma was collected from male Sprague Dawley blood samples. The blood samples were collected intravenous and centrifuged at 2000 rpm for 15 min in heparin coated vials. The supernatant was stored in aliquots at −20 °C. Radioactivity was measured with radio HPLC or in a gamma-counter (Wallac Wizard, PerkinElmer, Waltham, MA, USA). The determination of the distribution coefficient at pH 7.4 (log D_7.4) has been previously published [38 (link)].

Biodistribution of ¹¹¹In-CHX-DTPA-scFv78-Fc was also obtained in 8–12-week-old RD-ES tumor-bearing female common gamma KO Balb/c mice. Mice were injected intravenously with 150 kBq (range 30–440 kBq) ¹¹¹In-CHX-DTPA-scFv78-Fc and sacrificed 4 h (n = 3 mice), 24 h (n = 5 mice), 48 h (n = 4 mice), and 96 h (n = 3 mice) post-injection. Before injection, 25 μg of unlabeled scFv78-Fc were added to a saline solution containing 0.2 μg of ¹¹¹In-CHX-DTPA-scFv78-Fc, in order to administer the same amount of total antibody used for microPET studies. Mice were bled by cardiac puncture and sacrificed by cervical dislocation under anesthesia with isoflurane (2% in 1 L/min medical air). Organs were excised and weighed, and radioactivity was counted on a gamma counter (Wallac Wizard, Perkin Elmer, Waltham, MS, USA). For each time point, the activity of each source organ of each single animal was normalized by the total injected activity to obtain nA. For each source organ at each time point, an average nA value was obtained ±1SD.