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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

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Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

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, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD

Created: ; Last Update: June 4, 2009.

Chemical name:Cy5.5-Rituximab
Abbreviated name:Cy5.5-RIT
Agent category:Antibody
Target category:Antigen
Method of detection:Optical, near-infrared (NIR) fluorescence
Source of signal:Cy5.5
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about CD20.



Optical fluorescence imaging is increasingly used to obtain biological functions of specific targets in small animals (1, 2). However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350–700 nm) are used. Near-infrared (NIR) fluorescence (700–1,000 nm) detection avoids the background fluorescence interference of natural biomolecules, providing a high contrast between target and background tissues. NIR fluorophores have wider dynamic range and minimal background as a result of reduced scattering compared with visible fluorescence detection. They also have high sensitivity, resulting from low infrared background, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is becoming a non-invasive alternative to radionuclide imaging.

CD20 is a nonglycosylated phosphoprotein specific for the B-cell lymphocytes, and it plays an important role in B-cell development (3). Its expression is absent in the pro-B-cells and plasma B-cells, the first stage and the last stage of B-cell differentiation, respectively. CD20 is expressed on the cell surface of B-cell lymphoma, hairy cell leukemia, and B-cell chronic lymphocytic leukemia (4). Rituximab (RIT), a humanized monoclonal antibody (mAb) directed toward CD20, is approved by the United States Food and Drug Administration to treat B-cell lymphoma and leukemia (5). It is also under investigation in several clinical trials for combination treatments with chemotherapy (6). RIT has been conjugated with Cy5.5 (Cy5.5-RIT) to study in vivo biodistribution of Cy5.5-RIT in tumor-bearing mice (7). Cy5.5-RIT has been found to have a good accumulation in lymphoma tumor cells in nude mice. Cy5.5 is a NIR fluorescence dye with an absorbance maximum at 675 nm and an emission maximum at 694 nm, with a high extinction coefficient of 250,000 M-1cm-1.



RIT (6.6 nmol) was conjugated with Cy5.5-N-hydroxysuccinimide (NHS) ester (0.05 mg) to form Cy5.5-RIT after incubation for 1 h at room temperature (7). The NHS ester of Cy5.5 reacted with the ε-amino group of the lysine moieties of RIT. Cy5.5-RIT was purified with dialysis. There were three Cy5.5 dye molecules per RIT molecule. The labeling yield was 46%.

In Vitro Studies: Testing in Cells and Tissues


Biffi et al. (7) performed flow cytometric analysis with Cy5.5-RIT and RIT using the CD20-expressing human BJAB lymphoma cell line. Binding of Cy5.5-RIT and unlabeled RIT to the BJAB cells were similar, indicating that the immunoreactivity of Cy5.5-RIT was similar to the parent mAb. Control FITC-IgG1 was not able to bind the BJAB cells.

Animal Studies



Biffi et al. (7) performed whole-body NIR fluorescence imaging studies of Cy5.5-RIT in nude mice bearing subcutaneous BJAB lymphoma xenografts. Images were obtained after intravenous injection of 1 nmol of the tracer. Substantial contrasts were observed between the BJAB tumor and normal tissue from ~4 h with a maximal difference at 48 h. The NIR fluorescence intensity of the tumor slowly decreased over the following 8 days. The liver showed the fastest initial accumulation of the probe with gradual washout. Ex vivo imaging showed that most of the Cy5.5-RIT NIR fluorescence intensity was found in the tumor, with the liver exhibiting the second highest signal intensity; low signal intensities were observed in the kidneys, heart, lung, and spleen. Immunohistochemical staining showed that strong membrane binding to anti-CD20 antibody in the tumor was colocalized with RIT binding in the periphery of the primary tumor. However, no blocking experiment was performed.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.


Ntziachristos V., Bremer C., Weissleder R. Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging. Eur Radiol. 2003;13(1):195–208. [PubMed: 12541130]
Achilefu S. Lighting up tumors with receptor-specific optical molecular probes. Technol Cancer Res Treat. 2004;3(4):393–409. [PubMed: 15270591]
Reff M.E., Carner K., Chambers K.S., Chinn P.C., Leonard J.E., Raab R., Newman R.A., Hanna N., Anderson D.R. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood. 1994;83(2):435–45. [PubMed: 7506951]
McLaughlin, P., C.A. White, A.J. Grillo-Lopez, and D.G. Maloney, Clinical status and optimal use of rituximab for B-cell lymphomas. Oncology (Williston Park), 199812(12): p. 1763-9; discussion 1769-70, 1775-7. [PubMed: 9874849]
Cheson B.D. Monoclonal antibody therapy for B-cell malignancies. Semin Oncol. 2006;33(2) Suppl 5:S2–14. [PubMed: 16720198]
Tsirigotis P., Economopoulos T. Monoclonal antibodies in the treatment of lymphoid malignancies. J Steroid Biochem Mol Biol. 2008;108(3-5):267–71. [PubMed: 17935974]
Biffi S., Garrovo C., Macor P., Tripodo C., Zorzet S., Secco E., Tedesco F., Lorusso V. In vivo biodistribution and lifetime analysis of cy5.5-conjugated rituximab in mice bearing lymphoid tumor xenograft using time-domain near-infrared optical imaging. Mol Imaging. 2008;7(6):272–82. [PubMed: 19123997]
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