Peptides and antibodies and conjugation with DTPA or DOTA
DTPA-Tyr3-octreotide, DOTA-Tyr3-octreotide, [Lys40(DTPA)]exendin-3 [DTPA-exendin-3] and [Lys40(DOTA)]exendin-3 [DOTA-exendin-3]  were purchased from Peptide Specialty Laboratories GmbH (Heidelberg, Germany). The chimeric mAb anti-CAIX (cG250) was obtained from Wilex AG (Munich, Germany). The conjugation of anti-CAIX with SCN-Bz-DTPA or SCN-Bz-DOTA (Macrocyclics, Dallas, TX, USA) with a 50-fold molar excess was performed in a 0.1 M NaHCO3 buffer, with a pH of 8.2. After 1-h incubation, the conjugation mixture was dialyzed in a dialysis cell with a molecular cut-off value of 20 kD (Slide-a-lyzer, Pierce, Rockford, IL, USA) against 0.25 M ammonium acetate (pH 5.5) with five buffer changes to remove the unconjugated SCN-Bz-DTPA and SCN-Bz-DOTA. After conjugation, the protein concentration was determined spectrophotometrically (Amersham Pharmacia Biotech, Uppsala, Sweden) at 280 nm. The substitution ratio was determined by the labelling of the conjugation mixture with 111InCl3 (Covidien, Petten, The Netherlands) described by Hnatowich et al. . After incubation at room temperature [RT] for 20 min, quality control was performed on silica-gel instant thin layer chromatography [ITLC] strips (ITLC-SG, Biodex Medical Systems, Inc., Shirley, NY, USA) with sodium citrate, with a pH of 5.5, as the mobile phase (retention factor [R
f] 111In-labelled anti-CAIX mAb = 0, R
111In-DTPA or 111In-DOTA = 1). The substitution ratio is represented by the percentage of activity with an R
f of 0 when the conjugation mixture is labelled.
Sodium acetate (Merck, Darmstadt, Germany) was dissolved in distilled water (Versol, Lyon, France) to a final concentration of 0.1 M, and the pH was adjusted to 5.5 by titration with 1 M HCl (Merck, Darmstadt, Germany). Ammonium acetate buffer was prepared by mixing equal volumes of 0.2 M acetic acid (Merck, Darmstadt, Germany) and 0.2 M ammonia (Merck, Darmstadt, Germany), and the pH was adjusted to 5.5 by adding 0.2 M acetic acid or 0.2 M ammonia. MES and HEPES (Sigma-Aldrich Corporation, St. Louis, MO, USA) were dissolved in distilled water to a final concentration of 0.1 M, and the pH was adjusted to 5.5 with 1 M NaOH (Merck, Darmstadt, Germany).
111In-labelling of peptides and antibodies
The labelling of the six compounds with 111In was performed 9 days after 111In production (The calibration date of 111InCl3 is 10 days after the production of 111InCl3, and the expiry date of 111InCl3 is 11 days after the production of 111InCl3). The peptides and antibodies were dissolved in metal-free water to a final concentration of 0.1 μg/μl, and 5 μl was added to a 0.1 M NaAc, NH4Ac, MES or HEPES buffer. Five volumes of buffer and one volume of 111InCl3 (Covidien, Petten, The Netherlands) were added. The reaction mixtures were incubated for 20 min at RT for DTPA-conjugated compounds, at 95°C for DOTA-exendin and DOTA-octreotide or at 45°C for the DOTA-conjugated anti-CAIX antibody. After incubation, Tween80 (Sigma-Aldrich Corporation, St. Louis, MO, USA) was added to a final concentration of 0.1%, and ethylenediaminetetraacetic acid [EDTA] (Sigma-Aldrich Corporation, St. Louis, MO, USA) in 0.25 M NH4Ac, with a pH of 5.5, was added to a final concentration of 5 mM to complex unincorporated 111In. Quality control was performed on silica-gel ITLC strips with 0.1 M EDTA in 0.1 M NH4Ac as a mobile phase (R
111In-labelled compounds = 0, R
111In-EDTA = 1). The maximum SA was determined by correcting the initial SA for the radiochemical purity.
Effect of ageing of the 111InCl3 stock on the labelling efficiency of DTPA-exendin-3
DTPA-exendin-3 (0.5 μg) was labelled in triplicate (except for t = 14, which is in duplicate) with 111In (75 MBq) in 0.1 M NaAc, NH4Ac, MES and HEPES, with a pH of 5.5, as described above, from 4 days after the production date (delivery of 111InCl3) until 14 days after the production date of 111InCl3. Quality control was performed as described above.
Effect of the presence of cadmium on the labelling efficiency of DTPA-exendin-3
The effect of cadmium, the decay product of 111In, on the radiolabelling was examined by adding increasing amounts of Cd2+ to the labelling mixture of DTPA-exendin-3. CdCl2 (Sigma-Aldrich Corporation, St. Louis, MO, USA) was dissolved in 0.1 M Ultrapure HCl (J.T. Baker, Deventer, The Netherlands), and serial dilutions ranging from 10-1 to 10-7 M CdCl2 in 0.02 M HCl were prepared. DTPA-exendin-3 (0.5 μg) was labelled with 1.85 MBq 111InCl3 (at day 9 after 111InCl3 production) in 0.1 M NaAc, NH4Ac, MES and HEPES, with a pH of 5.5, as described above, and various amounts of CdCl2 were added simultaneously with 111InCl3 to amounts ranging from 1 × 10-3 to 9 × 104 nmol (resulting in final concentrations of Cd2+ ranging from 1 pM to 8.3 μM). The amount of buffer was adjusted for the amount of CdCl2 in 0.02 M HCl added (final pH 5.5). The experiment was performed in triplicate for all CdCl2 concentrations and all buffers. Quality control was performed as described above.