833c&Freso, 833cX&Freso, and fresolimumab were radiolabeled with iodine-125 (125I) (PerkinElmer, Waltham, MA) using Pierce™ Iodination Beads (Thermo Fisher Scientific, Carlsbad, CA) according to the manufacturer’s instructions, resulting in specific activities ranging from 5.5μCi/μg to 13μCi/μg. Radiolabeling did not affect apparent Kd values as determined by ELISA. SPECT/CT imaging was performed using a nanoScan SPECT/CT imaging system (Mediso, USA). Rats were injected iv with the indicated amount of antibodies 24 h after bleomycin treatment, and SPECT/CT images were captured after 1 h and 24 h. Note that the 1 h timepoint is an average, because the imaging process takes about 30 to 90 min. Images were acquired for 60 sec/projection using a four head gamma camera with a rat-seized multi-pinhole. Images were collected in a 360° orbit with 60 s sampling every 6°. The pulse height analyzer window was >28.40 keV with a width of 20%. SPECT/CT reconstruction was performed using Nucline nanoScan software (Mediso, Arlington, VA). CT voltage was set at 50 Kv, exposure for 300 msec and 720 helical projections were captured. After CTSPECT fusion, SPECT/CT 3-D data sets were processed with VivoQuant software (Invicro, Boston, MA). SPECT Planar imaging with 125I-833c&Freso was done at 3 μg/animal.
Pierce iodination beads
Manufactured by Thermo Fisher Scientific
Sourced in United States, Sweden, Japan
Pierce Iodination Beads are a solid-phase iodination reagent designed for the labeling of proteins and other biomolecules with radioactive iodine. The beads contain immobilized lactoperoxidase, which catalyzes the iodination reaction.
Automatically generated - may contain errors
Lab products found in correlation
Na125i, by PerkinElmer (2 mentions)
Iodine 125 125i, by PerkinElmer (1 mentions)
Nucline nanoscan software, by Mediso (1 mentions)
Vivoquant software, by Invicro (1 mentions)
Nanoscan spect ct imaging system, by Mediso (1 mentions)
Zeba micro spin desalting column, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 nhs ester, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Wallac 1470 wizard gamma counter, by PerkinElmer (1 mentions)
7k mwco zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
Zetasizer nano zsp, by Malvern Panalytical (1 mentions)
Zeba spin desalting columns 7k mwco 0.5 ml, by Thermo Fisher Scientific (1 mentions)
Sulfo nhs, by Merck Group (1 mentions)
2470 wizard2 gamma counter, by PerkinElmer (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Sodium phosphate dibasic, by Thermo Fisher Scientific (1 mentions)
Sodium phosphate monobasic monohydrate, by Thermo Fisher Scientific (1 mentions)
Mouse laryngoscope, by Penn-Century (1 mentions)
Bent fine dissecting forceps, by Thermo Fisher Scientific (1 mentions)
Sodium acetate, by Thermo Fisher Scientific (1 mentions)
10 protocols using pierce iodination beads
1
In Vivo Imaging of Radiolabeled Antibodies in Lung Injury Model
2
Labeling and Biodistribution of SOD
3
Radiolabeling and Pharmacokinetics of Peptides
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Peptides were labeled with iodine 125 (I125) radioisotope (PerkinElmer, Waltham, MA, USA; product no. NEZ033H005MC) using typical Pierce iodination beads (Thermo Scientific, Waltham, MA, USA; cat. no. 28665) according to the manufacturer’s protocol. In brief, the total amount of beads needed for the reaction was determined based on the amount of peptide to be labeled (1 bead was added to the reaction for every 50 µg of peptide). After washing with phosphate-buffered saline (PBS) and drying, the I125 solution was incubated with the beads for 5 minutes. The measured peptide solution in PBS was then added and allowed to react for 15 to 20 minutes. The beads were then separated from the reaction vials using long forceps, and the I125-labeled peptides were dialyzed through ultracentrifuge filters (1K cutoff) to separate free from bound I125. Labeling efficiency was determined by thin-layer chromatography with proper eluent (85% methanol).
Radiolabeled peptides were injected into the mouse tail vein and blood was collected from the opposite tail vein 5 minutes, 30 minutes, 60 minutes, 3 hours, and 24 hours after intravenous administration of labeled peptide. The blood was weighed and radioactivity was measured using a gamma counter. Radioactivity was normalized to the weight of the collected blood (activity/g) and plotted to determine the blood half-life.
Radiolabeled peptides were injected into the mouse tail vein and blood was collected from the opposite tail vein 5 minutes, 30 minutes, 60 minutes, 3 hours, and 24 hours after intravenous administration of labeled peptide. The blood was weighed and radioactivity was measured using a gamma counter. Radioactivity was normalized to the weight of the collected blood (activity/g) and plotted to determine the blood half-life.
4
Antibody-SOD Conjugate Preparation
5
Radiolabeled Rat IgG Conjugation to Nanoparticles
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Rat IgG (ThermoFisher catalog #31933) was labeled with I-125 via Pierce Iodination Beads (ThermoFisher catalog # 28665). We then conjugated the IgG to nanoparticles using our published protocol[15 (link)]. In brief, 100 μL of polystyrene NPs were buffer exchanged with Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL (ThermoFisher catalog # 89882), exchanging for 50 mM MES buffer at pH 5.2, finally putting the buffer-exchanged beads into 1.5 mL Eppendorf tubes. Next N-Hydroxysulfosuccinimide (“sulfo-NHS”; Sigma catalog #56485) was added to a final concentration of 0.275 mg/ml and incubated for 3 minutes at room temperature (RT). Next N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (“EDAC”; Sigma catalog # E7750) was added to a final concentration of 0.1 mg/ml and incubated for 10–15 minutes at RT. Next 114 ug of I-125-labeled rat IgG was added (giving 200 antibody molecules per bead) and incubated for 2 to 4 hours at RT on a vortex/shaker at low speed. 1 mL MES buffer was added to dilute free antibody, followed by centrifuge at 12,000g x 3 min to pellet the IgG-conjugated NPs. The IgG-NP pellet was resuspended in 200 uL of PBS + 0.05% bovine serum albumin (BSA) buffer. Immediately before use, the NPs were sonicated with a probe / tip sonicator (Qsonica Q55 Sonicator, Cole-Parmer #UX-04712-51) for three 3-second pulses at 30% maximum power.
6
Antibody-Conjugated Nanoparticle Synthesis
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NPs
prepared with PF127-maleimide and PF127-biotin were coated with Ab-SATA
and Ab-SA, respectively. NPs and modified antibodies were mixed for
5 min at room temperature (longer times did not show enhanced antibody
conjugation, unpublished data). As mentioned above, a Sepharose CL-4B
gel filtration column was used for removal of free drug and antibody.
Binding efficiency was measured by radiotracing a 10% substitution
of 125I-IgG-SATA or 125I-IgG-SA. A t test (two-tail, homoscedastic) was used to compare conjugation chemistries;
a p-value of 0.05 was considered statistically significant.
Antibody radiolabeling was performed using iodination beads as
instructed by the manufacturer (Pierce Iodination Beads, Thermo Scientific,
Pittsburgh, PA). The extent of radiolabeling was measured using a
standard trichloroacetic acid (TCA) assay. Two microliters of labeled
antibody, 1 mL of 3% BSA, and 200 μL of TCA were mixed and incubated
at room temperature for 15 min. Following centrifugation (15 min,
4 °C, 2300g), the amount of free iodine in the
supernatant was quantified using a Wizard2 2470 gamma counter
(PerkinElmer; Waltham, MA).
prepared with PF127-maleimide and PF127-biotin were coated with Ab-SATA
and Ab-SA, respectively. NPs and modified antibodies were mixed for
5 min at room temperature (longer times did not show enhanced antibody
conjugation, unpublished data). As mentioned above, a Sepharose CL-4B
gel filtration column was used for removal of free drug and antibody.
Binding efficiency was measured by radiotracing a 10% substitution
of 125I-IgG-SATA or 125I-IgG-SA. A t test (two-tail, homoscedastic) was used to compare conjugation chemistries;
a p-value of 0.05 was considered statistically significant.
Antibody radiolabeling was performed using iodination beads as
instructed by the manufacturer (Pierce Iodination Beads, Thermo Scientific,
Pittsburgh, PA). The extent of radiolabeling was measured using a
standard trichloroacetic acid (TCA) assay. Two microliters of labeled
antibody, 1 mL of 3% BSA, and 200 μL of TCA were mixed and incubated
at room temperature for 15 min. Following centrifugation (15 min,
4 °C, 2300g), the amount of free iodine in the
supernatant was quantified using a Wizard2 2470 gamma counter
(PerkinElmer; Waltham, MA).
7
Iodination and Purification of Exosomes
8
Radiolabeling of Hyaluronic Acid Derivatives
9
Labeling Albumin with Radioactive Iodine
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L-ring-2H5 phenylalanine, 99 atom percent (Cambridge Isotope Laboratories Inc., Andover MA, USA), was dissolved in sterile water together with unlabeled phenylalanine to a concentration of 20 mg/ml, 10 molar percent excess. The solutions were prepared, filter-sterilized, and stored in sterile 100-ml containers by APL Pharma Specials (Huddinge, Sweden).
Commercial 125I-labeled albumin 185 kBq/ml was purchased (Seralb-125©; IBA molecular, Gif-sur-Yvette Cedex, France). 123I-labeled albumin was prepared in the Department of Nuclear Medicine at Karolinska University Hospital, Huddinge, Sweden by incubation of sodium iodine (123I, 37 MBq/ml; Mallinckrodt Medical) with Albunorm 200 mg/ml (Octapharma, Stockholm, Sweden) and Pierce Iodination Beads (product number 28665; Thermo Fisher Scientific Inc., Waltham, MA, USA), and then filtering through a gel column for separation of free iodine (product number 17-0851-01, PD-10 Desalting column; Amersham Biosciences, Uppsala, Sweden). The final preparation contained less than 2 % free iodine.
Commercial 125I-labeled albumin 185 kBq/ml was purchased (Seralb-125©; IBA molecular, Gif-sur-Yvette Cedex, France). 123I-labeled albumin was prepared in the Department of Nuclear Medicine at Karolinska University Hospital, Huddinge, Sweden by incubation of sodium iodine (123I, 37 MBq/ml; Mallinckrodt Medical) with Albunorm 200 mg/ml (Octapharma, Stockholm, Sweden) and Pierce Iodination Beads (product number 28665; Thermo Fisher Scientific Inc., Waltham, MA, USA), and then filtering through a gel column for separation of free iodine (product number 17-0851-01, PD-10 Desalting column; Amersham Biosciences, Uppsala, Sweden). The final preparation contained less than 2 % free iodine.
10
Iodination and Characterization of Bovine Casoxin M
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BCM was purchased from Sigma. Di-amino acid peptide (tyr-pro; 1st and 2nd amino acids of BCM) was synthesized by Peptide Institute Inc. (Ibaraki, Japan). BCM and the tyr-pro peptide were labeled by chloramine-T method with 125I (J-RAM, Tokyo, Japan) using Pierce™ iodination beads (Thermo Fisher). Labeled BCM was incubated with CD10 in neutral peptidase assay buffer (40 mM Tris-HCl pH 7.1, 4 mM MgCl2, 0.5 mM ZnCl2) for 16 h. Thereafter, the solution was incubated with anti-BCM antibody (Abnova, Taipei City, Taiwan) at 37 °C for 2 h. The solution was subjected to immunoblot analysis in 20–25% sodium dodecyl sulfate-polyacrylamide gradient gels (Multigel II, Cosmo Bio, Tokyo, Japan). The radioactivity of the solution was also measured by a fluid scintillation counter (LSC-8000, Hitachi, Tokyo, Japan).
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