Badar, A;
DeFreitas, S;
McDonnell, JM;
Yahya, N;
Thakor, D;
Razavi, R;
Smith, R;
... Mullen, GED; + view all
(2011)
Recombinant Complement Receptor 2 Radiolabeled with [Tc-99m(CO)(3)](+): A Potential New Radiopharmaceutical for Imaging Activated Complement.
PLOS ONE
, 6
(4)
, Article e18275. 10.1371/journal.pone.0018275.
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Abstract
We describe the design and synthesis of a new Tc-99m labeled bioconjugate for imaging activated complement, based on Short Consensus Repeats 1 and 2 of Complement Receptor 2 (CR2), the binding domain for C3d. To avoid non specific modification of CR2 and the potential for modifying lysine residues critical to the CR2/C3d contact surface, we engineered a new protein, recombinant CR2 (rCR2), to include the C-terminal sequence VFPLECHHHHHH, a hexahistidine tag (for site-specific radiolabeling with [Tc-99m(CO)(3)(OH2)(3)](+)). The protein was characterized by N-terminal sequencing, SDS-PAGE and size exclusion chromatography. To test the function of the recombinant CR2, binding to C3d was confirmed by enzyme-linked immunosorbent assay (ELISA). The function was further confirmed by binding of rCR2 to C3d(+) red blood cells (RBC) which were generated by deposition of human or rat C3d and analyzed by fluorescence microscopy and flow cytometry. The affinity of rCR2 for C3d(+), in presence of 150 mM NaCl, was measured using surface plasma resonance giving rise to a K-D approximate to 500 nM. Radiolabeling of rCR2 or an inactive mutant of rCR2 (K41E CR2) or an unrelated protein of a similar size (C2A) with [Tc-99m(CO)(3)(OH2)(3)](+) at gave radiochemical yields >95%. Site-specifically radiolabeled rCR2 bound to C3d to C3d(+) RBC. Binding of radiolabeled rCR2 to C3d was inhibited by anti-C3d and the radiolabeled inactive mutant K41E CR2 and C2A did not bind to C3d(+) RBCs. We conclude that rCR2-Tc-99m has excellent radiolabeling, stability and C3d binding characteristics and warrants in vivo evaluation as an activated complement imaging agent.
| Type: | Article |
|---|---|
| Title: | Recombinant Complement Receptor 2 Radiolabeled with [Tc-99m(CO)(3)](+): A Potential New Radiopharmaceutical for Imaging Activated Complement |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1371/journal.pone.0018275 |
| Publisher version: | http://dx.doi.org/10.1371/journal.pone.0018275 |
| Language: | English |
| Additional information: | © 2011 Badar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The authors acknowledge financial support from King's College London, Division of Imaging Sciences, MRC Centre of Transplantation, The Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy's & St Thomas' NHS Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. |
| Keywords: | DELAYED GRAFT FUNCTION, RENAL-TRANSPLANTATION, REPERFUSION INJURY, ESCHERICHIA-COLI, ACUTE REJECTION, C3, MICE, INFLAMMATION, INHIBITOR, ISCHEMIA |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/1305226 |
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