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Serum stability and non-specific binding of technetium-99m labeled diaminodithiol for protein labeling
Affiliation:1. Sydney School of Public Health, The University of Sydney, Sydney, Australia;2. Centre for Kidney Research, The Children’s Hospital at Westmead, Sydney, Australia;3. College of Medicine and Public Health, Flinders University, Adelaide;4. Department of Nephrology, Princess Alexandra Hospital, Faculty of Medicine, University of Queensland, Brisbane, Australia;5. Department of Pediatrics, BC Children''s Hospital and University of British Columbia, Vancouver, Canada;6. Child and Adolescent Renal Service, Lady Cilento Children’s Hospital and University of Queensland, Brisbane, Australia;7. Pediatrics-Renal, Texas Children’s Hospital/Baylor College of Medicine, Houston, TX;8. Department of Nephrology, Royal Children’s Hospital Melbourne, University of Melbourne and Murdoch Children’s Research Institute, Victoria Australia;9. Department of Pediatrics, University of Melbourne and Murdoch Children’s Research Institute, Victoria Australia;10. Selzman Institute for Kidney Health, Section of Nephrology, Baylor College of Medicine, Houston, TX;11. UCL Centre for Nephrology and Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom;12. Department of Pediatrics and Child Health, The Children’s Hospital Research Institute of Manitoba, University of Manitoba, Winnipeg, Canada;13. Departments of Pediatrics and Epidemiology, Perelman School of Medicine, Division of Nephrology, The Children’s Hospital of Philadelphia, Philadelphia, PA;14. Department of Pediatrics, School of Medicine, University of Michigan, Ann Arbor, MI;15. Division of Nephrology and Hypertension, Cincinnati Children''s Hospital Medical Center, Cincinnati, OH;16. Department of Pediatric Nephrology, Emma Children''s Hospital AMC Academic Medical Center, Amsterdam, the Netherlands;17. Department of Pediatrics, Section of Nephrology, University of Calgary, Calgary, Canada;18. Division of Nephrology, Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India;19. Department of Pediatric Nephrology and NIHR/Wellcome Trust Clinical Research Facility, University of Manchester, Manchester Academic Health Science Centre, Royal Manchester Children’s Hospital, Manchester, United Kingdom;20. Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore;21. Department of Pediatrics, Division of Nephrology, Toronto Hospital for Sick Children, University of Toronto, Toronto, Canada;1. Laboratoire Lagrange, Université Côte d’Azur, Observatoire de la Côte d’Azur, CNRS, CS 34229, 06304 Nice Cedex 4, France;2. JHU/APL, USA;3. ESA/ESAC, Spain;4. Ondrejov Obs., Czech Republic;5. TU Braunschweig, Germany;6. Open University, UK;7. Royal Observatory of Belgium, Belgium;8. Aristotle University of Thessaloniki, Greece;9. Max-Planck Institute, Göttingen, Germany;10. German Aerospace Center, DLR, Cologne, Germany;11. UVSQ (UPSay), UPMC (Sorbonne Univ.), CNRS/INSU, LATMOS-IPSL, Guyancourt, France;12. Univ. Grenoble Alpes, IPAG, CNRS, IPAG, Grenoble, France;13. ESA/Hq, Paris, France;14. JPL, Pasadena, USA;15. Univ. Maryland, College Park, USA;p. Lowell Observatory, Flagstaff, USA;q. Universidad de Alicante, Spain;1. Department of Chemistry, 2 Plymouth Close, The University of the West Indies, Mona Campus, Kingston 7, Jamaica;2. Division of Chemistry, 127 Old Hope Road, University of Technology, Kingston 6, Jamaica;4. Department of Chemistry, Georgetown University, Washington, DC 20057, United States
Abstract:Previously we investigated the use of DTPA-coupled proteins to simplify labeling with 99mTc but especially to improve the stability of the label. These investigations have now been extended to include several N2S2 ligands such as N,N′-bis(2-methyl-2-mercaptopropyl)ethylenediamine (DADT) and a novel ligand of similar structure with a propylene bridge between two amines, 2-hydroxy-N,N′-bis(2-methyl-2-mercaptopropyl)propylenediamine (DADT-3C-2OH). The condition of labeling of free ligand (pH, buffer and tin concentration) was optimized to provide 100% chelation with 99mTc at reasonable ligand concentrations (100 μg/mL or less). Labeling was determined by paper chromatography, reverse-phase and size-exclusion HPLC. After incubation in fresh serum, 37 °C for 24 h, repeat analysis showed less than 5% dissociation of the chelate. By contrast, the DTPA chelate shows instability towards oxidation during this period. DADT derivatized on an ethylene carbon showed almost identical serum stability as DADT itself whereas when derivatized on a nitrogen greater instabilities were apparent. Using identical labeling conditions, free DADT was chelated in the presence of IgG at different ligand: protein molar ratios. Non-specific binding of 99mTc to IgG at a 10:1 DADT-HM:IgG molar ratio was as little as 5% and was essentially zero at a 2:1 DADT:IgG molar ratio when labeling was by transcomplexation from 99mTc-EDTA. The DADT-3C-2OH ligand showed superior performance both in regard to serum stability and the absence of non-specific binding. In conclusion, the N2S2 ligands form more stable chelates with 99mTc than does DTPA with reduced non-specific binding and may therefore represent an attractive alternative for labeling proteins with 99mTc by the bifunctional chelate approach.
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