89Zr radiolabeled ICOS antibody (rat clone 7E.17G9, Bio X cell) was prepared as previously described.12 (link) Briefly, 1 mg of antibody was diluted to 1 mg/mL in PBS, and the pH was adjusted to 8.8 to 9.0 prior to addition of deferoxamine-isothiocyanate (DFO-SCN) chelate (Macrocyclics) dissolved in DMSO (Thermofisher). The bioconjugation reaction was allowed to proceed for 1 hour at 37°C, after which the DFO-modified mAb was washed using a 2 mL Vivaspin filter with a 50 kDa cutoff (Sartorius) to remove unbound chelate. Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry was conducted on the bioconjugates to determine final chelate:mAb ratios (1.5-2.5 chelates per antibody), and final protein concentrations were determined using a Thermo Scientific NanoDrop UV-Vis spectrophotometer. For radiolabeling, ∼1 mCi 89Zr oxalate (3D Imaging), adjusted to pH 7.1 to 7.8 using 1 M Na2CO3, was incubated with the DFO-ICOS mAb for 1 hour at 37°C with gentle agitation. Free 89Zr oxalate was removed, and 89Zr-DFO-ICOS mAb was purified using 7K MW cutoff zeba spin desalting column (Thermofisher) and centrifuged for 1 minute at 1000g. Final radiochemical purity of >99% of 89Zr-DFO-ICOS mAb was determined using instant thin-layer chromatography using chromatography strips (Biodex).
Chromatography strips
Manufactured by Mirion Technologies
Sourced in United States
Chromatography strips are a type of laboratory equipment used for the separation and analysis of complex mixtures. They are designed to separate individual components within a sample based on their relative affinities for a stationary and mobile phase. The core function of chromatography strips is to facilitate the separation and identification of substances within a mixture.
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4 protocols using chromatography strips
1
Radiolabeling of ICOS Antibody with Zirconium-89
2
Radiolabeling Procedure for ABY-025
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Labelling was performed as described previously (23 (link)). In short, 50 µg of ABY-025 was diluted in 50 µl 0.2 M ammonium acetate buffer (pH 5.3), mixed with 50 MBq 111InCl (Medtronic, Minneapolis, MN, USA) and incubated at 60°C for 40 min. Labelling yield was determined on chromatography strips (Biodex Medical Systems, Shirley, NY, USA) in 0.2 M citric acid and analysed in a Phosphor Imager (Cyclone Storage Phosphor System; PerkinElmer, Inc., Waltham, MA, USA).
3
Encapsulation Efficiency of 57Co-Chelated Lipid Nanoparticles
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For the radioactive ion, 57 Co, the encapsulation efficiency of chelated OEP- 57 Co and OEP- 57 Co-OA inside NPs were evaluated by TLC. 57 Co (5.7 MBq) was chelated with 25 nmol OEP and then with
different amounts of OA. The LNP (200 µM) were then formulated with OEP- 57 Co or OEP- 57 Co-OA and the encapsulation efficiency was measured by TLC using chromatography strips (Biodex), mounted with 5 µL sample and eluted with 0.2 M citric acid (pH 2) and cut at the line. The radioactivity of both halves was measured in an Atomlab 950 well counter calibrated with Cs-137.The free 57 Co eluted to the top of the TLC strip while OEP- 57 Co and OEP- 57 Co-OA were retained in the NP and did not elute and so was measured at the bottom of the TLC strip as described above. All measurements were carried out in triplicate.
different amounts of OA. The LNP (200 µM) were then formulated with OEP- 57 Co or OEP- 57 Co-OA and the encapsulation efficiency was measured by TLC using chromatography strips (Biodex), mounted with 5 µL sample and eluted with 0.2 M citric acid (pH 2) and cut at the line. The radioactivity of both halves was measured in an Atomlab 950 well counter calibrated with Cs-137.The free 57 Co eluted to the top of the TLC strip while OEP- 57 Co and OEP- 57 Co-OA were retained in the NP and did not elute and so was measured at the bottom of the TLC strip as described above. All measurements were carried out in triplicate.
4
Microwave-Assisted Synthesis of Cobalt-Oleic Acid Complex
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The main challenge was to stably incorporate a very hydrophilic Co 2+ ion, (logP: 0. (25 nmol) were dissolved in 1 mL of ethanol in a sealed glass vial, and the solution was placed in a microwave oven for 3 minutes at 850 W to produce the OEP-Co complex.
After the solution was cooled down to room temperature, OA (100 nmol i.e., a mol ratio of 4:1 with OEP-Co, unless otherwise specified) was added to the solution and the resulting mixture was placed again in the microwave oven for another 1 min at 850 W (Scheme 1).The resulting OEP-Co-OA complex was then used without further purification. (ranging from 0 to 5µM). The mixture was heated in a microwave for 3 min at 850W and the UV-Vis spectra were recorded in a Shimadzu UV-2600 spectrophotometer. All measurements were carried out in triplicate at 20 °C.
The chelation efficacy of 57 Co was evaluated by instant thin layer chromatography (TLC) using chromatography strips (Biodex), mounted with the 5 µL sample and eluted with 0.2 M citric acid (pH 2) and cut at the line. The radioactivity of both halves was measured in an Atomlab 950 well counter calibrated with Cs-137. Free cobalt eluted on top while OEP- 57 Co-OA was retained at the bottom.
After the solution was cooled down to room temperature, OA (100 nmol i.e., a mol ratio of 4:1 with OEP-Co, unless otherwise specified) was added to the solution and the resulting mixture was placed again in the microwave oven for another 1 min at 850 W (Scheme 1).The resulting OEP-Co-OA complex was then used without further purification. (ranging from 0 to 5µM). The mixture was heated in a microwave for 3 min at 850W and the UV-Vis spectra were recorded in a Shimadzu UV-2600 spectrophotometer. All measurements were carried out in triplicate at 20 °C.
The chelation efficacy of 57 Co was evaluated by instant thin layer chromatography (TLC) using chromatography strips (Biodex), mounted with the 5 µL sample and eluted with 0.2 M citric acid (pH 2) and cut at the line. The radioactivity of both halves was measured in an Atomlab 950 well counter calibrated with Cs-137. Free cobalt eluted on top while OEP- 57 Co-OA was retained at the bottom.
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