Microspin g 50

An amount of 25 µL of either ^99mTc complexes (C1–C4) or distilled water as a blank was incubated with fresh human plasma (475 µL) in a water bath at 37 °C. At 30 and 60 min, 25 µL samples were seeded in a size-exclusion chromatography column (Microspin G-50, GE Healthcare, Buckinghamshire, UK). Columns were centrifuged at 4000 rpm for 1 min, and the activity of the eluate and the column was measured with a solid scintillation counter. The protein-bound tracer was calculated as the percentage of activity eluted from the column.

The free fraction in vitro was determined by adding the respective radioligand (100 kBq) to plasma or CSF (200 µL) and mixed vigorously. Aliquots of 20–50 µL were transferred to size exclusion chromatography columns (MicroSpin G-50; GE Healthcare GmbH, Solingen, Germany, centrifuged for two min at 2000 rpm, and the radioactivity in the filtrate and in the resin were measured in the gamma-counter. Three independent experiments were performed in triplicates.
For the ex vivo determination of the radioligands binding to proteins the respective tracer (15 MBq) was administered intravenous to male Sprague Dawley rats (200–250 g) under isoflurane anesthesia. Blood samples were drawn intravenous after 5, 15 and 30 min and centrifuged for 5 min at 2000 rpm in heparin-coated vials. An aliquot of 50 µL was taken from the supernatant and added to an equal volume of water (10% TFA). The protein precipitate was centrifuged off for 5 min at 25,000 rpm and analyzed together with a further 50 µL aliquot of the supernatant in the gamma-counter. Two independent experiments were performed.

Serum protein binding was determined using Sephadex G-50 (GE Healthcare Vienna, Austria) size exclusion chromatography [38 (link)]. Aliquots (50 µL, n = 3) of the radioligand solution (~10 µM) were incubated in 450 µL freshly prepared human serum or 450 µL PBS (controls) and were kept at 37 °C. After 1, 2, and 4 h aliquots (25 µL) were directly transferred to the column (MicroSpin G-50, GE Healthcare) and after centrifugation (2 min, 2000 rcf) the column containing the free conjugate and the eluate containing the protein-bound conjugate were measured in the gamma counter. The percent of activity in both fractions was calculated thereafter.

For this procedure antifungal siderophore conjugates were labeled as described before and diluted with PBS to a concentration of approximately 9 µM. Next, 50 µL of this solution was added to 450 µL of PBS (control) or 450 µL of fresh human serum and incubated at 37 °C for 30, 60, and 120 min. At each timepoint, 25 µL of PBS/serum was analyzed by size exclusion chromatography using MicroSpin G-50 columns (Sephadex G-50, GE Healthcare, Vienna, Austria) according to the manufacturer’s protocol. Hereafter, the column and eluate were measured separately in the gamma counter and calculated by dividing measured counts of eluate by total counts and multiplied by 100, resulting in percentage of protein bound conjugate. Radioactivity in the eluate reflects the protein bound fraction and column bound, free-labeled siderophore conjugate (n = 3, three technical replicates).

The ³²P label was introduced at the 5′-end of an oligonucleotide (10 pmol) using T4 polynucleotide kinase (5 U, Thermo Fisher Scientific, USA) and (γ-³²P)ATP (0.4 MBq) in a buffer consisting of 50 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, and 5 mM DTT at 37°C for 30 min. Labeled oligonucleotides were purified on columns MicroSpin G-50 (GE Healthcare, USA). Radioactivity was measured on a Tracor Analytic Delta 300 (ThermoQuest/CE Instruments, USA).

Protein binding abilities of [⁶⁸Ga]TAFC, [⁶⁸Ga]TPFC, [⁶⁸Ga]DABuFC, and [⁶⁸Ga]FSC(suc)₃ were studied by incubating radiotracers in human serum at 37 °C for 30, 60, and 120 min. At selected time points, 25 μl of serum aliquots was applied on size exclusion spin columns MicroSpin™ G-50 (GE Healthcare, Vienna, Austria). Columns were centrifuged at 2000 rcf for 2 min to separate protein bound radiotracer (eluate) from non-protein bound radiotracer (column). Protein binding ability was determined by measuring activities of column and eluate in the gamma counter. The results were displayed as % protein bound to total activity applied (n = 3).

Radiolabelled compound was diluted with PBS to 1 mL (~9 µM) and 50 µL of that solution was added to 450 µL serum or 450 µL PBS as a control. After 30, 60 and 120 min incubation at 37 °C, aliquots of 25 µL were analysed by size exclusion chromatography using MicroSpin G-50 columns (Sephadex G-50, GE Healthcare, Vienna, Austria) according to the manufacturer’s protocol. Hereafter, column and eluate were measured separately in the gamma counter to calculate percentage of free- (column bound) and protein-bound (eluate) fraction.