Wizard 3 1480 automatic gamma counter

The complexation of copper was performed according to a procedure adapted from Fagadar-Cosma et al. [46 (link)]. In a 1.5 mL glass crimp vial, a blend of Cu(II)acetate and TPP (0.2 µmol, each) in 1 mL of ethanol underwent reflux at 85 °C for six hours using a Heidolph MR Hei-Standard hot plate (Heidolph Instruments, Kelheim, Germany) equipped with an aluminum heating block. Successful complexation was confirmed through UV/Vis spectroscopy by comparing the spectrum to the spectrum of a reference sample of commercial Cu-TPP.
The complexation of ⁶⁴Cu was achieved via an adapted procedure. An amount of 0.4 µmol TPP in 250 µL THF was added to the dry [⁶⁴Cu]CuCl₂ precipitate, obtained as described above. The activity was 3.25 MBq ⁶⁴Cu, corresponding to 356 fmol ⁶⁴Cu. The mixture was refluxed at 75 °C for 3 h in a 1.5 mL glass crimp vial. Successful complexation was confirmed by adding 10 µL of the reaction mixture to a 250 µL:250 µL two-phase mixture of octanol and water. After 15 min of mixing, a microliter syringe was used to sample 100 µL of each phase, and the ⁶⁴Cu activity was measured with a WIZARD 3” 1480 automatic gamma counter (Perkin Elmer, Waltham, MA, USA).

IP was assessed using ⁵¹Cr-EDTA assay, as described by Bjarnason and Peters [8 (link)], with only minor modifications.
After an overnight fast, a test solution of 0.37 MBq (10 μCi) of ⁵¹Cr-EDTA diluted in 10 mL of tap water was given to each subject. Urine was collected for the next 24 hours. Food and drinks were allowed ad libitum 30 min later with the exception of coffee, tea and alcoholics.
Two samples (3 mL) of the pooled 24-hour urine collection were counted, in a gamma counter system (Perkin Elmer; Wizard 3, 1480 automatic gamma counter), together with a 3 mL sample of a 1/50th of the orally administered dose. The estimated radiation dose received during the test was <0.01 mSieverts (effective dose equivalent), which is within the variations of natural background radiation [8 (link), 9 (link)].
The absorption of ⁵¹Cr-EDTA was expressed as a percentage of the orally administered dose that was excreted in the urine during 24 hours, using the following formula: [(average urinary count x urinary volume) x (standard counts x 50)] - 1.
The passage of high molecular weight ⁵¹Cr-EDTA into the bloodstream is directly related to the level of IP and is poorly affected by bacterial degradation in the case of small intestinal bacterial overgrowth. In normal subjects, 1% to 3% of the orally administered dose of ⁵¹Cr-EDTA gets absorbed from the gastrointestinal tract [10 (link)].

In vivo uptake of ⁸⁹Zr-DFO-MPA1 was evaluated in multiple solid (LNCaP-AR, BT474) and liquid tumor (K562, THP-1, BLCL) models with varying degree of PRAME expression. The radiotracer was administered by tail-vein injection (130–180 μCi, 50 μg of pAb in 100 μL of sterile saline; t = 0 hours) and 4–5 animals were euthanized by CO₂ asphyxiation at 4, 24, 48 and 72 hours post injection (p.i.). An additional time-point (120 hours p.i.) was added to the study in K562 xenografts (n=5). BLCL mice (n = 4) served as PRAME negative liquid tumor model and were euthanized at 72 hours post injection. To further evaluate in vivo targeting specificity a group of THP-1 mice (n = 4) were co-administered with a 20-fold excess of unlabeled MPA1 and euthanized at 24 hours p.i. 14 tissues were collected from all animals, rinsed in water, dried in air, weighted and counted on a Wizard 3 1480 automatic gamma counter (PerkinElmer Inc., Waltham, MA, USA) for accumulation of ⁸⁹Zr-radioactivity (counts per minute, CPM). The count data were background and decay corrected and compared to the total administered dose; expressed as percentage of injected dose per gram (%IA/g, mean ± SD).

Unlabeled (non-radioactive) MPA1 antibody was serially diluted in RPMI 1640 with 1% BSA, from 900 nM to 0.03 nM in triplicate set of vials. The unlabeled antibody dilutions were transferred to vials containing 1 × 10⁶ THP-1 cells each. ⁸⁹Zr-DFO-MPA1 (1 μCi) in 50 μl of RPMI 1640, 1% BSA, was added to each vial. The vials containing cells, unlabeled antibody and radiolabeled antibody were incubated for 1 hour at 4°C. The cells were then harvested by centrifugation (5 min, 1400 g, 4°C) and washed in cold washing buffer (50mM Tris, 150mM NaCl, pH 7.2). The remaining cell-bound radioactivity was measured on a Wizard 3 1480 automatic gamma counter (PerkinElmer Inc., Waltham, MA, USA). Three unharvested vials were counted and served as standards. Non-specific binding was measured by harvesting and counting three vials incubated without cells; the average CPM was subtracted from the data, which was expressed as cell/media CPM. The data was fit to a single-site isotherm (nonlinear regression) model using Prism 7 GraphPad software (GraphPad Inc., San Diego, CA, USA) and an IC₅₀ was calculated.