The kinase IGF-1 receptor (KIRA) assay

The KIRA assay was used to assess residual ligand bioactivity in the circulation of IGF-TRAP-treated mice and performed as described in detail elsewhere^19,29 including recent modifications³⁰. In brief, HEK293 cells overexpressing IGF-1R were cultured in complete DMEM medium (containing 1% Penicillin/Streptomycin, 0.5 mg/ml G418, 0.25 mg/ml Hygromycin, 10% FCS). The cells were then seeded in a 48-well microtiter plate (4 × 10⁵ cells per well) for 24 h with serum, followed by a 24 h incubation without serum. After 48 h, sera from mice injected with 0.5 or 10 mg/kg IGF-TRAP diluted (1:10) in Krebs Ringer buffer or serially diluted rhIGF-1 (concentrations ranging from 0.3125–10 ng/ml, for standard curve) were added to the cells. Following a 15 min incubation at 37 °C to activate the IGF-1R, the cells are lysed (in HEPES lysis buffer) and the lysate transferred to a microtiter plate pre-coated with an IGF-1R capture antibody (Human phospho-IGF-1R ELISA kit, R&D Systems, Minneapolis, MN) and probed with an HRP-conjugated anti-phospho-tyrosine antibody. IGF-1R activation levels were measured at a wave length of 450 nm. Area under the IGF-1 serum concentration vs time curves up to day 18 were calculated using Phoenix WinNonlin Software v8.0.0.3176 (Pharsight Corporation).