Itlc sg

The iFAP freeze-dried kit was reconstituted with a [^99mTc]TcO₄Na/0.2 M phosphate buffer (1:1 v/v, 2 mL, 740 MBq) solution, and after incubation in a block heater (92 °C, 15 min), the radiochemical purity (R.P.) of the [^99mTc]Tc-iFAP radioligand was greater than 98%, as revealed by reversed-phase HPLC analysis (Discovery C18 column, particle size of 5 µm, 2.5 cm length, 0.46 cm I.D.; Supelco, Millipore, Burlington, MA, USA) applying the following linear gradient: flow rate of 1 mL/min, 0.1% TFA/water (A) (from 100 to 50%, over 10 min, maintained for 10 min, 30% over 5 min, and returned to 100% over 5 min), 0.1% TFA/acetonitrile (B).
ITLC-SG (Instant thin layer chromatography-silica gel) strips (Pall Corporation, Port Washington, NY, USA) were also used for the evaluation of the [^99mTc]Tc-iFAP R.P. The mobile phases were the following: (1) 2-butanone (used for the identification of free [^99mTc]TcO₄Na (Rf = 1), where [^99mTc]Tc-iFAP, the radiocolloid, and [^99mTc]Tc-EDDA/tricine remained in the origin (Rf = 0)), (2) sodium citrate (0.1 M, pH 5), which identified free [^99mTc]TcO₄Na (Rf = 1) and [^99mTc]Tc-EDDA/tricine(Rf = 1), while [^99mTc]Tc-iFAP and the radiocolloid remained in the origin (Rf = 0), and (3) methanol-ammonium acetate (1:1 v/v), used as the phase for the determination of the radiocolloid (Rf = 0) (Rf = 1 for [^99mTc]TcO₄Na, [^99mTc]Tc-iFAP, and [^99mTc]Tc-EDDA/tricine).

Untagged #2F8 and R3B23 (3 mg/mL) reconstituted in 50 mM sodium carbonate buffer pH 8.5 were reacted with a tenfold molar excess of bifunctional chelator CHX-A’’-DTPA (Macrocyclics) for 3 h at RT, as described previously [22 (link)]. The conjugation reaction was quenched by reducing the pH of the mixture to pH 7.0. DTPA-2F8 was purified on Superdex 75 10/300 GL (GE Healthcare) in 0.1 M ammonium acetate buffer pH 7.0. The necessary amount of ¹¹¹In (54 to 270 MBq) or ¹⁷⁷Lu (74 MBq to 1 GBq) was incubated for 30 min at RT or 37 °C, respectively, with the DTPA-2F8 in 200 mM ammonium acetate pH 5. Resulting ¹¹¹In- and ¹⁷⁷Lu-DTPA-2F8 were purified on NAP-5 column (GE Healthcare) using 0.9% NaCl as eluent. In case of high radioactive labelling for preclinical therapy, 0.9% NaCl with 5 mg/mL ascorbic acid was used. The resulting radio-conjugates were filtered on 0.22 μm after which RCP was determined by iTLC with citric acid as running buffer (iTLC-SG, Pall Corporation) and measured > 96%.

Nanobodies were labeled with [^99mTc(H₂O)₃(CO)₃]⁺ at their His6-tag via tricarbonyl chemistry, as described previously (38 (link)). The ^99mTc-labeled nanobodies were purified from the unbound [^99mTc(H₂O)₃(CO)₃]⁺via NAP-5 SEC (Sephadex, GE-Healthcare, Chicago, USA), and filtered through a 0.22 µm filter (Millex, Millipore, Burlington, USA). The radiochemical purity of radiolabeled nanobodies was evaluated by instant thin layer chromatography-silica gel (iTLC-SG, Pall Corporation, Belgium).