The antibodies were conjugated on their lysine residues using p-SCN-Bn-CHX-A”-DTPA (Macrocyclics, Plano, TX, USA) [39 (link)]. Briefly, the antibodies were dialyzed against 1L 0.05 M HEPES, pH 8.5 containing 5 g Chelex 100 (Bio-Rad, Hercules, CA, USA) using Maxi GeBAflex-tube dialysis tubes (MWCO 8 kDa, Gene Bio-Application, Israel). CHX-DTPA was then added in 5-fold molar excess and reacted for 2 h at room temperature. Conjugation solutions were purified by dialysis into 0.4 M ammonium acetate pH 5.5. 111In radiolabeling was carried out at 37° for 30 min at pH 5, and analysed by iTLC-SG (Agilent, Santa Clara, CA, USA). For tumor localization, C57BL/6 mice carrying DT6606 tumors were injected i.v. with radiolabeled antibodies and imaged at 24 and 48 h post injection by SPECT/CT (Bioscan, Santa Barbara, CA, USA.
P scn bn chx a dtpa
Manufactured by Macrocyclics
Sourced in United States
P-SCN-Bn-CHX-A"-DTPA is a chemical compound used in the field of laboratory equipment. It serves as a chelating agent, binding to metal ions to form stable complexes. The core function of this product is to facilitate the labeling and detection of biomolecules in various analytical and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Itlc sg, by Agilent Technologies (2 mentions)
Chelex 100, by Bio-Rad (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
Ar 2000, by Bioscan (1 mentions)
P scn bn nota, by Macrocyclics (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Nap 5 column, by GE Healthcare (1 mentions)
Cetuximab, by MedChemExpress (1 mentions)
Jetoptimus transfection reagent, by Polyplus Transfection (1 mentions)
Chelex, by Merck Group (1 mentions)
Chelex 100 sodium form, by Merck Group (1 mentions)
P scn bn dota, by Macrocyclics (1 mentions)
Sephadex g 50, by GE Healthcare (1 mentions)
Mouse igg, by Merck Group (1 mentions)
10 protocols using p scn bn chx a dtpa
1
Antibody Radiolabeling and Tumor Localization
2
Antibody-Mediated Growth Inhibition Assay
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Pellets were then resuspended in BronchiaLife media with Lifefactor without antibiotic and cell density measured. The suspension was then diluted so that 103Y. pestis cells could be plated into a 96-well plate at a final volume of 200 µL. Antibodies αF1Ig 2, αF1Ig 8, and αM2IgG were then added to each well at concentrations of 0.35–0.09 µM. The plate was incubated at 37°C into a Biotek NTX Plate reader (capable of shaking) and cell-density readings (OD600) were taken overnight every 10 minutes after 1 minute’s shaking to track bacterial growth. αF1Igs were conjugated with p-SCN-Bn-CHX-A”-DTPA (Macrocyclics, Plano, TX, USA), through solvent-exposed lysines' condensation to thiourea. Briefly, a 10 mg/mL solution of p-SCN-Bn-CHX-A”-DTPA was prepared in DMSO. The antibodies were buffer-exchanged into NaHCO3 (0.1 M, pH 8.5) using a Zeba Spin desalting column (Thermo Fisher Scientific), and a molar ratio of 10:1 p-SCN-Bn-CHX-A”-DTPA in DMSO was added. The reaction was allowed to proceed for 1 hour at 37°C with vigorous shaking. Excess p-SCN-Bn-CHX-A”-DTPA and salts were removed by a second buffer exchange into NH4CH3COO (0.1 M, pH 5.5) with a second Zeba Spin desalting column. The final concentration of antibody was determined with a Bradford assay.
3
Radiolabeling of Onartuzumab Conjugate
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Onartzumab stock buffer was exchanged with PD10 column into PBS and pH adjusted to 8.5 with 1 M Na2CO3. The bifunctional chelate, p-SCN-Bn-CHX-A"-DTPA (Macrocyclics, Plano, TX), resuspended in DMSO, and reacted with onartuzumab at a 10:1 ratio and incubated at 37ºC for 90 minutes. The construct was purified with PD10 column using 200 mM ammonium acetate (pH=5.5) containing 6 mg/mL ascorbic acid buffer for radiolabeling. Lu-177 that was obtained from ITM Isotope Technologies (Garching, Germany) and was incubated with purified onartuzumab-CHX-A'' conjugate at 37ºC for 60 minutes. Labeling was monitored using radio-iTLC with silica-gel impregnated glass-microfiber paper strips (iTLC-SG, Varian, Lake Forest, CA, analyzed using an AR-2000, Bioscan Inc., Washington, DC), eluted with a mobile phase of aqueous solution of EDTA (50 mM, pH 5.5). The product was purified using PD10 column equilibrated with 6 mg/mL ascorbic acid in PBS (pH=7) adjusted with 1M Na2CO3. Radiochemical purity was assessed by radio-iTLC as described above.
Details of characterization of the bioconjugates and radioconjugates including mass spectrometry analysis, serum stability, and pharmacokinetic profiles are described fully in theSupplemental Information
Details of characterization of the bioconjugates and radioconjugates including mass spectrometry analysis, serum stability, and pharmacokinetic profiles are described fully in the
4
Radiolabeling of Single-Domain Antibodies
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All commercially obtained chemicals were of analytical grade. Recombinant sdAb-proteins were produced without terminal tags by the Vlaams Instituut voor Biotechnologie (VIB) Protein Service Facility (VIB, Gent) in Pichia pastoris and were formulated in phosphate-buffered saline 1× (PBS) (0.01 M phosphate buffer/0.14 M NaCl) pH 7.4 during the final batch purification. p-SCN-Bn-NOTA and p-SCN-Bn-CHX-A″-DTPA were purchased from Macrocyclics (Macrocyclics, Inc., Plano, TX, USA). 68Ga was obtained from a 68Ge/68Ga Galli EoTM generator (IRE Elit, Fleurus, Belgium), indium chloride ([111In]InCl3) from Curium (Hertogenbosch, The Netherlands). High-purity water for trace analysis (TraceSELECT, Riedel-de-Haën, Honeywell Research Chemicals, Seelze, Germany) was used for the preparation of radiolabeling buffer.
5
Radiolabeling and Stability of Antibody Conjugates
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Conjugation, radiolabeling and stability studies were performed as previously described 18 . In summary, hu11B6 and non-specific isotype control IgG1 were conjugated with p-SCN-Bn-CHX-A”-DTPA (Macrocyclics, Dallas, TX), a backbone substituted diethylenetriaminepentaacetic acid (DTPA) derivate chosen as a trade-off between stability and metal ion complexation 19 (link), in a 1:3 molar ratio. 50 - 250 MBq 177LuCl3 (IDB Petten BV, Baarle-Nassau, Holland) was added to 150 µg of hu11B6 (~1 µg/µL) in ammonium acetate buffer (pH of ~5.5), with a final volume of 500 µL. After 2 h of incubation, free 177Lu was eluted from 177Lu-DTPA-hu11B6 ([177Lu]hu11B6) and 177Lu-DTPA-IgG1 ([177Lu]IgG1) using a NAP-5 column (GE Healthcare, Uppsala, Sweden) equilibrated with saline or PBS. Stability of [177Lu]hu11B6 was studied by incubating samples (n = 3) in mouse serum at 37°C for 1 w, using labeled conjugate in PBS as a control. Separation was performed on NuPAGE®Bis-Tris Gel (Life technologies via Thermo Fischer Scientific). The conjugation and radiolabeling of the non-specific antibody, an IgG antibody from mouse serum (Sigma, I-8765), was performed as above. Binding affinity of 177Lu-hu11B6 was evaluated and compared to non-labeled hu11B6 using Surface Plasmon Resonance (SPR), see suppl. information.
6
Radiolabeled scFv78-Fc Fusion Protein
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The scFv78-Fc fusion protein targeting endosialin/tumor endothelial marker 1 (TEM1) was obtained as previously described [15 (link)] and conjugated with 10 eq. of the chelator p-SCN-Bn-CHX-A”-DTPA (Macrocyclics, cat. no. B-355) at 42 °C and pH 9.1 (full details will be reported elsewhere). The final solution was diluted in 0.9% NaCl to give a concentration of 5 mg/mL CHX-DTPA-scFv78-Fc.
A solution of 15 MBq of 152Tb in 90 μL α-HIBA was added to 20 μL of CHX-DTPA-scFv78-Fc dissolved in 100 μL of NH4OAc buffer (pH 5.4, 0.4 M). After 1 h incubation at 42 °C, 152Tb-CHX-DTPA-scFv78-Fc was obtained without further purification with a purity > 95% (iTLC, citrate buffer pH 4.6).
For biodistribution experiments, a commercially available 111In chloride solution (200 MBq Mallinckrodt) was added to a mixture of NH4OAc buffer (pH 5.4, 0.4 M, 100 μL) and CHX-DTPA-scFv78-Fc (50 μL). After 1 h incubation at 42 °C, an 80–90% conversion was obtained (iTLC, citrate buffer pH 4.6). The product was diluted with 0.9% NaCl and purified by ultrafiltration.
A solution of 15 MBq of 152Tb in 90 μL α-HIBA was added to 20 μL of CHX-DTPA-scFv78-Fc dissolved in 100 μL of NH4OAc buffer (pH 5.4, 0.4 M). After 1 h incubation at 42 °C, 152Tb-CHX-DTPA-scFv78-Fc was obtained without further purification with a purity > 95% (iTLC, citrate buffer pH 4.6).
For biodistribution experiments, a commercially available 111In chloride solution (200 MBq Mallinckrodt) was added to a mixture of NH4OAc buffer (pH 5.4, 0.4 M, 100 μL) and CHX-DTPA-scFv78-Fc (50 μL). After 1 h incubation at 42 °C, an 80–90% conversion was obtained (iTLC, citrate buffer pH 4.6). The product was diluted with 0.9% NaCl and purified by ultrafiltration.
7
Cetuximab Radiolabeling Protocol
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Cetuximab (Cat# HY-P9905) was purchased from MedChemExpress (Monmouth, NJ, USA). The human IgG1 isotype (Cat# BE0297) was purchased from BioXCell (Lebanon, NH, USA) and used as a control. The jetOPTIMUS transfection reagent for transient transfection in cells was purchased from Polyplus (Alsace, Grand Est, France). The bifunctional chelating agent [(R)-2-Amino-3-(4-isothiocyanatephenyl) propyl]-trans-(S, S)-cyclohexane-1,2-diamine-pentaacetic acid (p-SCN-Bn-CHX-A-DTPA, Cat# B-355) was purchased from Macrocyclics Inc. (Richardson, TX, USA).
8
Radiolabeling Chelators DTPA, DOTA, NETA
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Reagents, unless specified otherwise, were purchased from Sigma-Aldrich (Bornem, Belgium) and used without further purification. Solvents were purchased from VWR (Leuven, Belgium) or Sigma-Aldrich, and used without further purification. p-SCN-Bn-CHX-A”-DTPA (DTPA ) and p-SCN-Bn-DOTA (DOTA ) were purchased from Macrocyclics, Inc. (Texas, USA), and p-NCS-Bz-DOTA-GA (DOTA-GA ) was purchased from CheMatech (Dijon, France), and used without further purification. 3p-C-NETA-NCS (NETA ) was synthesized and characterized according to literature methods (19 (link)). All radiolabeling buffers were stirred with chelex [chelex 100 sodium form (50–100 mesh, Sigma Aldrich)], to remove trace metals, for 15 min and then filtered to remove the chelex beads. All solutions were degassed and filtered before use.
9
Radiolabeling of Mouse IgG with Indium-111
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IgG is the most common type of antibody found in blood circulation [24 (link)]. Mouse IgG (Sigma-Aldrich, St. Louis, MO, USA) was conjugated with p-SCN-Bn-CHX-A″-DTPA (Macrocyclics, Dallas, TX, USA), as previously described [25 (link)]. The p-SCN-Bn-CHX-A″-DTPA-conjugated antibody was purified using a Sephadex G-50 (GE Healthcare) column (700× g for 2 min). The conjugation ratio of chelate to antibody was estimated to be approximately 1.5, as determined by cellulose acetate electrophoresis. The chelate-conjugated antibody (0.5 nmol) was mixed with 1.48 MBq of 111InCl3 (Nihon Medi-Physics) in 0.5 M acetate buffer (pH 6.0) and the mixture was incubated for 30 min at room temperature. The radiolabeled antibody was separated from free In-111 using a Sephadex G-50 column (700× g for 2 min once). The labeling yields of 111In-IgG ranged from 71.4% to 86.5%, the radiochemical purity was 100%, and the specific activity was 14.1 to 17.1 kBq/μg. The approximate molecular weight of 111In-IgG was 147,111 Da.
10
Conjugation of Anti-PSCA mAb 7F5 with DTPA
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The anti-PSCA mAb 7F5 was modified with p-SCN-Bn-CHX-A″-DTPA (Macrocyclics, Plano, TX, USA) by coupling the functional isothiocyanate group to primary amines in the protein structure. First, the mAb was concentrated to about 1.0 mg/mL in a MWCO 30 kDa MacrosepTM centrifugal concentrator (PALL, Port Washington, NY, USA). Thereby, the buffer was exchanged with 0.05 M NaHCO3 (pH 6.4, containing 0.3 M NaCl) using the same centrifugal technique. For modification of 5 mg mAb, 4.4 mg p-SCN-Bn-CHX-A″-DTPA (185-fold molar excess) was dissolved in three times the volume 0.5 M HEPES (pH 7.2, containing 0.3 M NaCl) and added dropwise to the Ab solution. The mixture was incubated for 24 h at 23 °C with occasional slewing. The resulting conjugate was separated from excess of p-SCN-Bn-CHX-A″-DTPA and other reactants by repeated washing (6×) using, in total, 500 mL of 0.05 M HEPES (pH 6.0, containing 0.3 M NaCl) in a MWCO 30 kDa JumbosepTM centrifugal concentrator (Pall, Port Washington, NY, USA). SEC-HPLC of the conjugate was performed as described above for the unconjugated Ab.
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