Anti-CD20 Immunoglobulin G Radiolabeling with a 99mTc-Tricarbonyl Core: In Vitro and In Vivo Evaluations

PLoS One. 2015 Oct 6;10(10):e0139835. doi: 10.1371/journal.pone.0139835. eCollection 2015.


In recent years, the diagnostic and therapeutic uses of radioisotopes have shown significant progress. Immunoglobulin (Ig) appears to be a promising tracer, particularly due to its ability to target selected antigens. The main objective of this study is to optimize and assess an Ig radiolabeling method with Technetium 99m (99mTc), an attractive radioelement used widely for diagnostic imaging. Monoclonal anti-CD20 IgG was retained to study in vitro and in vivo radiolabeling impact. After IgG derivatization with 2-iminothiolane, IgG-SH was radiolabeled by an indirect method, using a 99mTc-tricarbonyl core. Radiolabeling stability was evaluated over 24h by thin-layer chromatography. IgG integrity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis coupled with Western blot and autoradiography. The radiolabeled Ig's immunoaffinity was assessed in vitro by a radioimmunoassay method and binding experiments with cells (EL4-hCD20 and EL4-WT). Biodistribution studies were performed in normal BALB/c mice. Tumor uptake was assessed in mice bearing EL4-hCD20 and EL4-WT subcutaneous xenografts. With optimized method, high radiolabeling yields were obtained (95.9 ± 3.5%). 99mTc-IgG-SH was stable in phosphate-buffered saline (4°C and 25°C) and in serum (37°C), even if important sensitivity to transchelation was observed. IgG was not degraded by derivatization and radiolabeling, as shown by Western blot and autoradiography results. 99mTc-anti-CD20 IgG-SH immunoaffinity was estimated with Kd = 35 nM by both methods. In vivo biodistribution studies for 48h showed significant accumulation of radioactivity in plasma, liver, spleen, lungs and kidneys. Planar scintigraphy of mice bearing tumors showed a significant uptake of 99mTc-anti-CD20 IgG-SH in CD20+ tumor versus CD20- tumor. Radiolabeling of derivatized IgG with 99mTc-tricarbonyl was effective, stable and required few antibody amounts. This attractive radiolabeling method is "antibody safe" and preserves Ig affinity for antigen, as shown by both in vitro and in vivo experiments. This method could easily be used with noncommercial IgG or other antibody isotypes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Anti-Idiotypic*
  • Antigens, CD20 / immunology*
  • Chromatography, Thin Layer
  • Female
  • Immunoglobulin G / immunology*
  • Mice
  • Mice, Inbred BALB C
  • Radioimmunodetection
  • Tissue Distribution


  • Antibodies, Anti-Idiotypic
  • Antigens, CD20
  • Immunoglobulin G
  • anti-IgG

Grant support

Some of the work presented here was supported in part by CORC (Comité d’Orientation de la Recherche sur le Cancer) Limousin. Mallinckrodt Medical for supply of IsoLink. F. Dalmay (INSERM UMR-S 1094 NET) for assistance with statistical analyses. M. Cogne for gift of EL4-WT and EL4-CD20 cells. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. B Cell Design Society provided support in the form of a salary for author AC, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the "author contributions" section.