An Improved Method for Conjugating Monoclonal Antibodies with <i>N</i>-Hydroxysulfosuccinimidyl DOTA

Lewis, Michael R.; Kao, Jim Y.; Anderson, Aaron S.; Shively, John E.; Raubitschek, Andrew

doi:10.1021/bc0000886

Cited by 110 publications

(126 citation statements)

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“…The absence of free thulium was verified by adding a small aliquot of this equilibrium mixture to a solution of 0.15 M acetate buffer, pH 5. Acetate (7) was obtained by the method of Lewis et al (19). Compound L1 (2.9 mg, 6.6 µmol) was dissolved in water (1.043 mL, 4 °C), and the pH of the solution adjusted to 5 with 15 µL of 1 M HCl.…”

Section: Preparation Of Thulium Complexesmentioning

confidence: 99%

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Labeling of Adenovirus Particles with PARACEST Agents

et al. 2008

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Recombinant adenovirus type 5 particles (AdCMVLuc) were labeled with two different bifunctional ligands capable of forming stable complexes with paramagnetic lanthanide ions. The number of covalently attached ligands varied between 630 and 1960 per adenovirus particle depending upon the chemical reactivity of the bifunctional ligand (NHS ester versus isothiocyanide), the amount of excess ligand added, and the reaction time. The bioactivity of each labeled adenovirus derivative, as measured by the ability of the virus to infect cells and express luciferase, was shown to be highly dependent upon the number of covalently attached ligands. This indicates that certain amino groups, likely on the surface of the adenovirus fiber protein where cell binding is known to occur, are critical for viral attachment and infection. Addition of 177 Lu 3+ to chemically modified versus control viruses demonstrated a significant amount of nonspecific binding of 177 Lu 3+ to the virus particles that could not be sequestered by addition of excess DTPA. Thus, it became necessary to implement a prelabeling strategy for conjugation of preformed lanthanide ligand chelates to adenovirus particles. Using preformed Tm 3+ -L2, a large number of chelates having chemical exchange saturation transfer (CEST) properties were attached to the surface residues of AdCMVLuc without nonspecific binding of metal ions elsewhere on the virus particle. The potential of such conjugates to act as PARACEST imaging agents was tested using an on-resonance WALTZ sequence for CEST activation. A 12% decrease in bulk water signal intensity was observed relative to controls. This demonstrates that viral particles labeled with PARACEST-type imaging agents can potentially serve as targeted agents for molecular imaging.

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Section: Preparation Of Thulium Complexesmentioning

confidence: 99%

“…L1 was obtained by removal of benzyl protective group by hydrogenolysis with H 2 and 10% palladium on carbon in ethanol. The N-hydroxysuccinimide ester of L1 was generated in situ according to a previously published method (19). Ligand 2 was synthesized using a previously published method (17).…”

Section: Synthesismentioning

confidence: 99%

Labeling of Adenovirus Particles with PARACEST Agents

et al. 2008

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“…Other DOTA derivatives have been devised for conjugation to peptide amino groups, such as succinimide DOTA derivatives (20) and isothiocyanato DOTA derivatives (21). Unnatural amino acid derivatives have also been developed to couple DOTA to peptidyl amines, such as p-NH 2 -phenylalanine (13) and diaminopropionic acid residues (15).…”

Section: Introductionmentioning

confidence: 99%

Peptidyl Molecular Imaging Contrast Agents Using a New Solid-Phase Peptide Synthesis Approach

Yoo

Pagel

2007

Bioconjugate Chem.

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A versatile method is disclosed for solid phase peptide synthesis (SPPS) of molecular imaging contrast agents. A DO3A moiety was derivatized to introduce a CBZ-protected amino group and then coupled to a polymeric support. CBZ cleavage with Et 2 AlCl/thioanisole was optimized for SPPS. Amino acids were then coupled to the aminoDOTA loaded resin using conventional step-wise Fmoc SPPS to create a product with DOTA coupled to the C-terminus of the peptide. In a second study, the DO3A moiety was coupled to a glycine-loaded polymeric support, and amino acids were then coupled to the amino-DOTA-peptide loaded resin using SPPS, to incorporate DOTA within the peptide sequence. The peptide-(Tm 3+ -DOTA) amide showed a PARAmagnetic Chemical Exchange Saturation Transfer (PARACEST) effect, which demonstrated the utility of this contrast agent for molecular imaging. These results demonstrate the advantages of exploiting SPPS methodologies through the development of unique DOTA derivatives to create peptide-based molecular imaging contrast agents.

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“…18 Conjugates were labeled with 64 Cu to a specific activity of 10 μCi/μg in 0.1 M ammonium citrate, pH 5.5, for 1 h at 43°C. 19 Diethylenetriaminepentaacetic acid (DTPA) was then added to a final concentration of 1 mM, and the reaction mixtures were let stand for 15 min at room temperature. The labeled mAbs were purified and exchanged into phosphatebuffered saline by gel-filtration spin column chromatography, using Bio-Spin 6 columns (Bio-Rad, Hercules, CA).…”

mentioning

confidence: 99%

“…The labeled mAbs were purified and exchanged into phosphatebuffered saline by gel-filtration spin column chromatography, using Bio-Spin 6 columns (Bio-Rad, Hercules, CA). 19 Specific activity of labeling was determined prior to purification and radiochemical purity was determined after purification, by SE-HPLC using a Waters (Milford, MA) Delta 600 chromatograph equipped with a manual Rheodyne injector, a Waters 2487 dual wavelength UV detector, a Packard (Downers Grove, IL) 500TR Flow Scintillation Analyzer with a GAMMA-C flow cell for 64 Cu, a Waters busSAT/IN analog-digital interface, and the Waters Millennium 32 software package. A Phenomenex (Torrance, CA) BioSep-SEC-S 3000 column (7.8 x 300 mm, 5 μm, 290 Å), an isocratic mobile phase of 100 mM NaH 2 PO 4 /0.05% NaN 3 , pH 6.8 and a flow rate of 1.0 mL/min were used.…”

mentioning

confidence: 99%