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99mTc-Labeled anti-receptor for advanced glycation endproducts polyclonal antibody F(ab’)2 fragments

99mTc-anti-RAGE pF(ab’)2
, PhD
National Center for Biotechnology Information, NLM, NIH

Created: ; Last Update: August 16, 2010.

Chemical name:99mTc-Labeled anti-receptor for advanced glycation endproducts polyclonal antibody F(ab’)2 fragmentsImage RAGE.jpg
Abbreviated name:99mTc-anti-RAGE pF(ab’)2
Synonym:99mTc-anti-RAGE F(ab’)2
Agent Category:Antibodies
Target:Receptor for advanced glycation endproducts (RAGE)
Target Category:Receptors
Method of detection:Planar imaging, single-photon emission computed tomography (SPECT)
Source of signal / contrast:99mTc
  • Checkbox In vitro
  • Checkbox Rodents
Structure of RAGE.



The 99mTc-labeled anti-receptor for advanced glycation endproducts (RAGE) polyclonal antibody F(ab’)2 fragment, abbreviated as 99mTc-anti-RAGE pF(ab’)2 or 99mTc-anti-RAGE F(ab’)2, is a radiotracer developed by Tekabe et al. for imaging atherosclerotic lesions by targeting highly expressed RAGE (1). RAGE is a 35-kDa transmembrane receptor of the immunoglobulin (Ig) superfamily (1, 2).

RAGE has one V domain, two C domains, one transmembrane domain, and one cytoplasmic tail. The V domain consists of two N-glycosylation sites and is responsible for extracellular ligand binding. RAGE exists in three forms: full-length, membrane-bound, and soluble (1-3). The soluble Isoform of the RAGE protein, which lacks the transmembrane and the signalling domain are thought to counteract the detrimental action of the full-length RAGE. Under physiological conditions, RAGE is expressed at low levels in a variety of cells in a regulated manner, but RAGE is highly expressed in a series of age- and diabetes-related chronic inflammatory diseases and cancer (4-7). The advanced glycation endproducts (AGEs) are the major ligands of RAGE. AGEs are a heterogeneous group of peptides and proteins derived from non-enzymatic glycosylation processes (2, 4). Large amounts of AGEs is formed through metabolism and aging, and this establishes a positive feedback cycle under pathological conditions such as diabetes (1, 3, 8). The interaction between AGEs and RAGE affects almost all types of cells and molecules and results in pro-inflammatory gene activation (7, 9). The pathological effects induced by the AGEs/RAGE interaction include increasing vascular permeability, inhibiting vascular dilation, inducing cytokine secretion, enhancing oxidative stress, and modulating cell response to exogenous growth factors. Understanding of the AGEs/RAGE interaction is crucial to develop new treatment regimens for age- and diabetes-related conditions and cancer (3, 9).

In the case of atherosclerosis, AGEs are formed at a high level (10). RAGE itself is expressed in nearly all cell types pertinent for the development and progression of atherosclerotic plaque. The AGEs/RAGE interaction leads to diabetic vascular complications and augments atherosclerotic plaque development and progression (7, 8). In a proof-of-concept study of whether the expression level of RAGE could be detected and thus used as a marker of atherosclerosis, Tekabe et al. developed an anti-RAGE polyclonal antibody F(ab’)2 fragment and demonstrated the feasibility of using 99mTc-anti-RAGE pF(ab’)2 for the noninvasive detection of RAGE in the atherosclerotic plaques in apolipoprotein E–null (ApoE–/–) mouse model (1). The major limitation to this study is that polyclonal antibody fragments and planar imaging modality were used (1). Polyclonal antibodies usually have less specificity and higher background signal compared with monoclonal antibodies. Planar imaging systems have a lower spatial resolution than tomographic imaging systems such as single-photon emission computed tomography.



Tekabe et al. described in detail the production and radiolabeling of the polyclonal antibody F(ab’)2 fragment (1). Briefly, the peptide Ac-NRRGKEVKSNYRVRVYQIC-amide on the basis of the V domain of RAGE was synthesized and injected into rabbits. The serum was retrieved, and the IgG was prepared and affinity-purified. The F(ab')2 fragments were generated with pepsin digestion of the intact antibody. 99mTc-Labeling of the fragments was performed with diethylenetriamine pentaacetic acid (DTPA) modification, followed by reaction with 99mTcO4- in SnCl2 in 0.1 N HCl. The specific activity of the radiotracer 99mTc-anti-RAGE pF(ab’)2 was 1.8 ± 0.34 MBq/µg (48.7 ± 9.3 µCi/µg) protein and the radiochemical purity was 95 ± 1.6%. The radiolabeling yield was not reported. Nonspecific control IgG was prepared from nonimmune rabbit serum, fragmented into F(ab')2, and coupled to DTPA for 99mTc labeling as described above.

In Vitro Studies: Testing in Cells and Tissues


Immunoreactivities of the intact antibody and antibody fragment were tested against soluble RAGE (1). The antibody concentration for the DTPA-modified F(ab')2 fragment, given 50% of maximum binding, was 0.9 µg/ml, which was equivalent to 9 × 10–9 mol/L or apparent affinity of 0.11 × 109 L/mol. The concentration for the unmodified anti-RAGE IgG was 0.8 µg/ml, which was equivalent to 8 × 10–9 mol/L or apparent affinity of 0.12 × 109 L/mol.

Animal Studies



Tekabe et al. measured the blood-pool clearance of 99mTc-anti-RAGE pF(ab’)2 in male C57BL/6 mice to determine the optimal time for imaging (1). The clearance was biexponential, with the half-life equal to 15 min for the first (fast) component and 7 h for the second (slower) component. Therefore, Tekabe et al. performed planar γ imaging at 4 h after tracer injection in atherosclerotic model of male ApoE–/– mice (n = 13). Corresponding wild-type male C57BL/6 mice (n = 4) on normal chow were used as controls.

Planar γ imaging showed focal uptake for the 99mTc-anti-RAGE pF(ab’)2 in the atherosclerotic lesions in the proximal aorta (n = 6 mice), but no uptake for the nonspecific IgG F(ab’)2 (n = 6 mice). Control C57BL/6 mice injected with 99mTc-anti-RAGE pF(ab’)2 also showed no localization of the radiotracer in the proximal aorta (no lesions at gross examination). There was a significant difference between the three groups of mice in terms of tracer uptake in proximal aorta relative to whole body (Friedman; χ2 = 12.83; P = 0.0016) (1). This value was 1.03% for 99mTc-anti-RAGE pF(ab’)2, 0.17% for nonspecific IgG F(ab’)2 in the experimental ApoE–/– mice, and 0.31% for 99mTc-anti-RAGE pF(ab’)2 in the control C57BL/6 mice at 4 h after injection. Ex vivo counting confirmed the difference for the uptake in the proximal aorta among the three groups of mice (χ2 = 9.613; P = 0.0082) (1). Aortic uptakes were 1.76% and 0.13% injected dose per gram tissue (ID/g) in the ApoE–/– mice receiving 99mTc-anti-RAGE pF(ab’)2 and nonspecific IgG F(ab’)2, respectively, and this value was 0.39% ID/g in the control C57BL/6 mice receiving 99mTc-anti-RAGE pF(ab’)2. In the ApoE–/– mice injected with 99mTc-anti-RAGE pF(ab’)2, a significant difference for the radiotracer uptake was also observed between thoracic organs (aortic lesions, heart (0.50% ID/g), and lungs (0.90% ID/g)) (χ2 = 8; P = 0.0183). Histology and immunohistochemical staining of the proximal aorta confirmed formation of atherosclerotic lesions and uptake of the radiotracer in the lesions expressing RAGE (1). In biodistribution studies at 5–6 h after injection, the highest uptake was observed in the subdiaphragmatic organs (particularly in the liver and spleen) for both 99mTc-anti-RAGE pF(ab’)2 and nonspecific IgG F(ab’)2 as observed on the in vivo scans (1).

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.


Tekabe Y., Li Q., Rosario R., Sedlar M., Majewski S., Hudson B.I., Einstein A.J., Schmidt A.M., Johnson L.L. Development of receptor for advanced glycation end products-directed imaging of atherosclerotic plaque in a murine model of spontaneous atherosclerosis. Circ Cardiovasc Imaging. 2008;1(3):212–9. [PubMed: 19808545]
Yan S.F., Ramasamy R., Schmidt A.M. The RAGE axis: a fundamental mechanism signaling danger to the vulnerable vasculature. Circ Res. 2010;106(5):842–53. [PMC free article: PMC2862596] [PubMed: 20299674]
Sourris K.C., Forbes J.M. Interactions between advanced glycation end-products (AGE) and their receptors in the development and progression of diabetic nephropathy - are these receptors valid therapeutic targets. Curr Drug Targets. 2009;10(1):42–50. [PubMed: 19149535]
Daroux M., Prevost G., Maillard-Lefebvre H., Gaxatte C., D'Agati V.D., Schmidt A.M., Boulanger E. Advanced glycation end-products: implications for diabetic and non-diabetic nephropathies. Diabetes Metab. 2010;36(1):1–10. [PubMed: 19932633]
Abe R., Yamagishi S. AGE-RAGE system and carcinogenesis. Curr Pharm Des. 2008;14(10):940–5. [PubMed: 18473843]
Fang F., Lue L.F., Yan S., Xu H., Luddy J.S., Chen D., Walker D.G., Stern D.M., Schmidt A.M., Chen J.X., Yan S.S. RAGE-dependent signaling in microglia contributes to neuroinflammation, Abeta accumulation, and impaired learning/memory in a mouse model of Alzheimer's disease. FASEB J. 2010;24(4):1043–55. [PMC free article: PMC3231946] [PubMed: 19906677]
Nienhuis H.L., Westra J., Smit A.J., Limburg P.C., Kallenberg C.G., Bijl M. AGE and their receptor RAGE in systemic autoimmune diseases: an inflammation propagating factor contributing to accelerated atherosclerosis. Autoimmunity. 2009;42(4):302–4. [PubMed: 19811283]
Walcher D., Marx N. Advanced glycation end products and C-peptide-modulators in diabetic vasculopathy and atherogenesis. Semin Immunopathol. 2009;31(1):103–11. [PubMed: 19347338]
Ramasamy R., Yan S.F., Schmidt A.M. RAGE: therapeutic target and biomarker of the inflammatory response--the evidence mounts. J Leukoc Biol. 2009;86(3):505–12. [PubMed: 19477910]
Tekabe Y., Luma J., Einstein A.J., Sedlar M., Li Q., Schmidt A.M., Johnson L.L. A novel monoclonal antibody for RAGE-directed imaging identifies accelerated atherosclerosis in diabetes. J Nucl Med. 2010;51(1):92–7. [PubMed: 20008983]
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