Wallac 1470 automated gamma counter

The ¹⁷⁷Lu-anti-CD55 antibody was prepared as previously described^{39 (link)} with minor modifications. Briefly, an anti-CD55-specific monoclonal antibody was incubated with a 50-fold molar excess of [(R)-2-Amino-3-(4-isothiocyanatophenyl) propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid (p-SCN-Bn-CHX-A”-DTPA,B-355; Macrocyclics) in 0.1 mol/L NaHCO₃ buffer (pH 8.2) and conjugated antibodies purified. The p-SCN-Bn-CHX-A”-DTPA-conjugated anti-CD55 antibody was labeled with ¹⁷⁷Lu (Lu-177 n.c.a.; ITG; half-life, 6.71 days) in 0.1 mol/L ammonium acetate buffer (pH 5.4) for 30 min at room temperature.
The immunoreactivity of ¹⁷⁷Lu-anti-CD55 was evaluated using the Lindmo assay as described previously^{40 (link),41 (link)}. Briefly, H460 cells (0 to 6.0 × 10⁶) were incubated with 0.074 MBq of ¹⁷⁷Lu-anti-CD55 for 1 hour. The cells were washed and binding specificity analyzed using Scatchard assays. Radioactivity was quantified using a Wallac 1470 automated gamma counter (PerkinElmer Life Science). Saturation binding analyses were performed as described previously^{42 (link)}. Blocking assays were performed by plating 1.5 × 10⁵ of the indicated cells in 24-well plates and incubating them with 0.074 MBq of ¹⁷⁷Lu-anti-CD55 in the presence of 50X unlabeled anti-CD55 antibody. Non-specific binding was quantified as described previously^{43 (link)}.

The immunoreactivity of ⁴⁷Sc-DTPA-cetuximab was evaluated using the Lindmo method and an in vitro cell-binding assay. Following the Lindmo method, cells (1–5 × 10⁶) were incubated with 0.185 MBq ⁴⁷Sc-DTPA-cetuximab for 1 h at 4 °C. The cells were washed thrice with cold PBS and centrifuged at 1000 rpm for 5 min. Radioactivity was quantified using a Wallac 1470 automated gamma counter (PerkinElmer Life Sciences). For the in vitro cell-binding assay, cells (1 × 10⁶) were incubated with 0.185 MBq ⁴⁷Sc-DTPA-cetuximab for 1 h at 4 °C in the presence of 10 × unlabeled cetuximab and IgG as the control. The cells were washed thrice with cold PBS and centrifuged at 1000 rpm for 5 min. Radioactivity was quantified using a Wallac 1470 automated gamma counter. All experiments were performed with triplicate samples.