To evaluate the amounts of trastuzumab conjugated to AuNRs, the following experiments were conducted. At first, trastuzumab radiolabeled with 111 In was prepared. The purified trastuzumab was mixed with p-SCN-Bn-DTPA (Macrocyclics, Plano, TX, U.S.A.) in phosphate buffer (pH: 8.6) at a 1 : 2 (trastuzumab : p-SCN-Bn-DTPA) reaction ratio, and the reaction concentration of trastuzumab was found to be 2.4 mg/mL. After 6 h of incubation at 37 °C, DTPA-trastuzumab (DTPA-Tra) was purified by using Amicon Ultra-4 (30 kDa) filtration and labeled with 111 InCl 3 in acetate buffer (0.1 M, pH: 6.0) at r.t., followed by reaction with AuNRs as described above. This crude sample was applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bullet PAGE One Precast Gel; Nacalai Tesque, Kyoto, Japan), and 111 In-Tra-AuNRs and 111 Intrastuzumab were separated. The gel was analyzed by autoradiography (FUJIFILM BAS-5000; FUJIFILM, Tokyo, Japan), and the signal intensities (SIs) of bands were quantified. After evaluating the radioactivity level of the separated regions of interest corresponding to Tra-AuNRs and free trastuzumab, the number of trastuzumab molecules conjugated to AuNRs was calculated according to the following equation; (moles of reacted trastuzumab) × (SIs of 111 In-Tra-AuNRs)/(SIs of 111 In-Tra-AuNRs + SIs of 111 In-trastuzumab)/(moles of AuNRs).
P scn bn dtpa
Manufactured by Macrocyclics
Sourced in United States
P-SCN-Bn-DTPA is a chelating agent used for the synthesis of radiopharmaceuticals. It is a bifunctional chelator that can be conjugated to various biomolecules, allowing for the radiolabeling of those compounds.
Automatically generated - may contain errors
Lab products found in correlation
Gdcl3, by Merck Group (2 mentions)
Chelex resin, by Bio-Rad (2 mentions)
Glass microfiber chromatography paper, by Agilent Technologies (2 mentions)
Barnstead nanopure purification system, by Thermo Fisher Scientific (2 mentions)
Crc 25r dose calibrator, by Mirion Technologies (2 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
111in incl3, by Nordion (2 mentions)
P scn bn dota, by Macrocyclics (2 mentions)
Sk n sh, by American Type Culture Collection (2 mentions)
111 in 111 in incl 3, by Macrocyclics (2 mentions)
Osca78, by Kerafast (2 mentions)
Slide a lyzer cassette, by Thermo Fisher Scientific (1 mentions)
Tryptic soy broth (tsb), by Merck Group (1 mentions)
Tryptic soy agar, by Merck Group (1 mentions)
Sulfosuccinimidyl 4 n maleimidomethyl cyclohexane 1 carboxylate sulfo smcc, by Thermo Fisher Scientific (1 mentions)
Copper 2 sulfate, by Thermo Fisher Scientific (1 mentions)
Meso 2 3 dimercaptosuccinic acid, by Merck Group (1 mentions)
Macs ms column, by Miltenyi Biotec (1 mentions)
Minimacs separator, by Miltenyi Biotec (1 mentions)
Trastuzumab herceptin, by Genentech (1 mentions)
21 protocols using p scn bn dtpa
1
Quantifying Trastuzumab Conjugation to AuNRs
2
Radiolabeling Antibodies via DTPA Conjugation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To prevent contaminants from disturbing radiolabeling, 1 mg of mIgG1 PD-L1-srt-his and rIgG2a WT antibodies were dialyzed against 5 L sterile PBS (metal-free) using Slide-a-Lyzer Cassettes (ThermoFisher Scientific). Subsequently, a 15 eq. of S-2-(4-Isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid (p-SCN-Bn-DTPA, Macrocyclics, B-305) and 1/10 reaction volume 1 M NaHCO3 in PBS, pH 5.5 were added to each antibody and incubated for 1 h at RT. Non-conjugated p-SCN-DTPA was removed from the reaction mixture by dialysis against 5 L 0.25 M NH4Ac (metal-free, pH 5.5) using Slide-a-Lyzer Cassettes (ThermoFisher Scientific). After dialysis, concentration in all samples was determined via spectrophotometer.
3
Oleic Acid-Coated Iron Oxide Nanoparticles Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Oleic acid-coated iron oxide nanoparticles (20-nm SPION) dispersed in chloroform were obtained from Ocean NanoTech (San Diego, CA). GdCl3, meso-2,3-dimercaptosuccinic acid (DMSA), tryptic soy agar (TSA), and tryptic soy broth (TSB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC), sodium L-ascorbate, and copper (II) sulfate were purchased from Thermo Scientific, Uppsala, Sweden. bis-MPA ammonium dendron, acetylene core, Generation 4 (G4) were obtained from Polymer Factory, Sweden AB. p-SCN − Bn − DTPA was obtained from Macrocyclics, USA. Tris [3-hydroxypropyltriazolylmethyl] amine (THPTA) was purchased from Click Chemistry Tools LLC. PD MiniTrapTM G-10 was purchased from GE Healthcare. MiniMACS separator and MACS MS column were purchased from Miltenyi Biotec (cat#130–042-102). All other chemicals and reagents were of reagent grade and used without further purification.
4
Radiolabeling Trastuzumab with Indium-111
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) or Thermo Fisher Scientific (Pittsburgh, PA, USA), unless otherwise specified. Aqueous solutions were prepared using ultrapure water (resistivity, 18 MΩ.cm) treated with Chelex resin purchased from Bio-Rad Laboratories, Inc. (Berkeley, CA, USA). p-SCN-Bn-DTPA was purchased from Macrocyclics, Inc. (Dallas, TX, USA). Indium-111 ([111In] InCl3) was purchased from MDS Nordion (Vancouver, BC, Canada). Trastuzumab (Herceptin), an anti-human HER-2 IgG monoclonal antibody was obtained from Genentech (San Francisco, CA, USA).
5
Radiolabeling Reagents Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All reagents were purchased from Sigma-Aldrich unless otherwise stated and were used without further purification. The chelating agents p-SCN-Bn-DFO and p-SCN-Bn-DTPA were purchased from Macrocyclics Inc. (Dallas, TX). Water was deionised using a Barnstead NANOpure purification system (Thermo Scientific) and had a resistance of >18.2 MΩ cm−1 at 25 °C. Protein concentration measurements were made on a ND-1000 spectrophotometer (NanoDrop Technologies, Inc.). Instant thin-layer chromatography (iTLC) was performed on glass microfiber chromatography paper (Agilent Technologies) and strips were analysed with a Bioscan AR-2000 radio-TLC scanner (Eckert & Ziegler). pH was determined using pH indicator paper (Merck Millipore). Radioactivity measurements were made using a CRC-25R dose calibrator (Capintec, Inc.).
6
Synthesis of AHNP-PEG Conjugates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HS-PEG-NH2 (average molecular weight: 5 kDa), fluorescein isothiocyanate isomer I (FITC) and cyanine5.5 NHS ester (Cy5.5) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti HER2/neu peptides, AHNP (sequence: FCDGFYACYMDV), AHNP-PEG, FITC-labeled AHNP-PEG (FITC-AHNP-PEG) were custom-made by AnaSpec (Fremont, CA, USA). The p-SCN-Bn-DTPA was obtained from Macrocyclics (Plano, TX, USA).
7
Radiolabeling and In Vivo Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) or Thermo Fisher Scientific (Pittsburgh, PA, USA), unless otherwise specified. Aqueous solutions were prepared using ultrapure water (resistivity, 18 MΩ cm). p-SCN-Bn-DTPA and p-SCN-Bn-DOTA were purchased from Macrocyclics, Inc. (Dallas, TX, USA). [111In]InCl3 was purchased from MDS Nordion (Vancouver, BC, Canada). 225Ac was produced from a 229Th source as described in [21 (link), 22 (link)]. The InVivoPlus anti-mouse PD-L1 Ab (anti-PD-L1-BC) was purchased from BioXCell (West Lebanon, NH, USA). Blood chemistry was determined using a Spotchem EZ Vet from Scil Animal Care Company (Gurnee, IL, USA).
8
Radiolabeling of DTPA-Conjugated Nanobodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
JVZ-007-c-myc-his was incubated with a 5-fold molar excess of p-SCN-Bn-DTPA (Macrocyclics) in 0.1 M sodium carbonate buffer (pH 9.5) for 2.5 h at room temperature. JVZ-007-cys was reduced with 1 mM 2-mercaptoethylamine-HCl in phosphate-buffered saline (PBS), 5 mM ethylenediaminetetraacetic acid for 90 min at 37°C. Reduced JVZ-007-cys was then incubated with 5 mM maleimide-DTPA for 2 h at 37°C. Conjugated Nanobodies were then dialyzed for 3 d in a Slide-A-Lyzer (3.5-kDa cutoff; Life Technologies) against 0.25 M ammonium acetate (NH 4 Ac), pH 5.5.
Nanobody-DTPA conjugates were labeled with 111 InCl 3 (Covidien) in 20 mM sodium acetate, pH 5.0, for 30 min at room temperature. Radioprotectants (3.5 mM ascorbic acid, gentisic acid, and methionine) were used to prevent radiolysis. Labeling efficiency was assessed by instant thin-layer chromatography using silica gel-coated paper (Varian Inc.) and 0.1 M citrate buffer, pH 5.0, as the mobile phase. After incubation, an excess of DTPA (final concentration, 0.15 mM) was added to complex free 111 InCl 3 .
Nanobody-DTPA conjugates were labeled with 111 InCl 3 (Covidien) in 20 mM sodium acetate, pH 5.0, for 30 min at room temperature. Radioprotectants (3.5 mM ascorbic acid, gentisic acid, and methionine) were used to prevent radiolysis. Labeling efficiency was assessed by instant thin-layer chromatography using silica gel-coated paper (Varian Inc.) and 0.1 M citrate buffer, pH 5.0, as the mobile phase. After incubation, an excess of DTPA (final concentration, 0.15 mM) was added to complex free 111 InCl 3 .
9
Radiolabeling of Human IgG
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human IgG (Nanogam®, Sanquin, Amsterdam, The Netherlands) was used as aspecific control. IgG was conjugated with p-SCN-Bn-DTPA (Macrocyclics, Dallas, TX, USA) as described earlier [40 (link)]. Radiolabeling was performed using indium-111 (111In) chloride (Mallinckrodt, Prague, Czech Republic).
Radiochemical purity of 111In-IgG labeling was checked by instant thin layer chromatography (ITLC) using 0.1 M citrate buffer pH 6.0 as eluent.
Radiochemical purity of 111In-IgG labeling was checked by instant thin layer chromatography (ITLC) using 0.1 M citrate buffer pH 6.0 as eluent.
10
Radiolabeling HER2-targeting Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The HER2-targeting mAbs used in this study were the commercially available trastuzumab (Herceptin®, Roche) and ICR12, developed at The Institute of Cancer Research, London [18 (link), 19 (link)]. The bifunctional chelator 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (pSCN-Bn-DTPA, Macrocyclics, US) was conjugated to ICR12 and trastuzumab. Additionally, 2-(4-isothiocyanatobenzyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (p-SCN-Bn-DOTA, Macrocyclics, US) was conjugated to trastuzumab. The immunoconjugates (50–80 μg) were radiolabelled with 111In (ca 42 MBq) (Perking Elmer, US) in acetate buffer (pH = 4). All reactions were performed as described previously [20 (link), 21 (link)]. The use of different radioimmunoconjugates does not affect the results of this study, as the primary aims were to investigate the feasibility and challenges of image quantification and dosimetry in pre-clinical studies, rather than to compare the radiotracers.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!