Avidin-gadolinium

[Avidin-Gd]

Background

[PubMed]

Biotin is a protein found in mammalian cells and is considered to be a vitamin because it has an important function in the cellular metabolism of lipids and carbohydrates. Avidin is a protein that is found primarily in the egg white or in bacteria (known as streptavidin if isolated from bacteria; Streptomyces avidinii is the source of this protein). Although the two proteins have different origins, they are known to have a very strong non-covalent interaction (discovered almost seven decades ago) with a dissociation constant (K_d) of 4 × 10^-14 M (1, 2). The avidin–biotin interaction has been described in detail by Wilchek et al. (3). Only harsh conditions (e.g., treatment with formaldehyde at high temperature) are known to break the avidin/streptavidin–biotin bond, and the released proteins cannot be reused for any other work due to deterioration of the samples (4). Because of their strong interaction, the avidin/streptavidin–biotin system has been extensively studied and is widely used after conjugation of either protein to other proteins, nucleic acids, or other matrices for research and diagnostic purposes, including imaging techniques (5-8).

Because of its overexpression, the human epidermal growth factor receptor 2 (HER2/neu) is an important target for the diagnosis, treatment, and prognosis of cancers such as those of the breast, lungs, etc (9, 10). As a result, humanized monoclonal antibodies (MAbs) labeled with different nuclides or imaging and contrast agents targeted toward HER2/neu have been used for the in vitro and in vivo imaging of cancers (11-13). Some humanized MAbs against HER2/neu alone or their conjugated derivatives are used, or are in development, as immunotherapeutic agents for the treatment of this ailment (14, 15). The avidin–biotin system has been used to visualize HER2/neu-positive tumors in transgenic mice by some investigators with magnetic resonance imaging (MRI) (16, 17). To achieve this, the investigators prelabeled the cells expressing HER2/neu, under in vitro and in vivo conditions, with a commercially available biotinylated mouse MAb. The biotinylated MAb was allowed to wash or clear out from the respective test systems, and this was followed by the administration of avidin conjugated to gadolinium (avidin-Gd) to the cells or animals. MRI was subsequently performed, and it was demonstrated that a high positive T1 contrast could be achieved under the in vitro and in vivo conditions used.

Synthesis

[PubMed]

The synthesis of avidin-Gd was performed as described by Artemov et al. using a modified procedure of Hnatowich et al. (13, 18). Briefly, 20-fold excess cyclic anhydride of diethylenetriaminepentaacetic acid (DTPA) was allowed to react with avidin for 24 h at 4°C. Excess low molecular weight reagents from the conjugated avidin (avidin-DTPA) were removed by centrifuge filtration through a device with a 10K cut-off limit. The avidin-DTPA was then reacted with Gd-(1-(2-naphthoyl)-3,3,3-trifluoroacetonate)₂ to obtain avidin-GdDTPA (avidin-GdDTPA) as described above. Avidin-GdDTPA was separated from excess low molecular weight compounds as detailed above. On average, 12.5 GdDTPA groups were reported to be attached to each avidin molecule.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

For in vitro studies, NT-5 cells expressing HER-2/neu were derived from spontaneous mammary tumors of female HER-2/neu transgenic mice (13). In the same study, mouse carcinoma EMT-6 cells that do not express HER-2/neu were used as controls. Expression of HER-2/neu by the two cell lines was confirmed with fluorescence-activated cell-sorting analysis using appropriate reagents (13). The cells were exposed to biotinylated anti-HER-2/neu MAb in phosphate-buffered saline (PBS) containing bovine serum albumin (BSA) for 30 min at room temperature. The cells were then washed with PBS several times and incubated with avidin-GdDTPA for 5 min at room temperature. Control cells were exposed to a non-specific MAb or to avidin-GdDTPA. The cells were washed again as detailed above and fixed in 2% paraformaldehyde in PBS in a nuclear magnetic resonance tube. Subsequently, T1-weighted images (repetition time, TR = 1 s; spin echo time, TE = 8 ms) were obtained with an MRI probe. Only the NT-5 cells that were exposed to the biotinylated anti-HER-2/neu MAb showed a positive T1 contrast with avidin-GdDTPA, and the EMT-6 cells had a low background signal.

Animal Studies

Human Studies

[PubMed]

No references are currently available.

Supplemental Information

[Disclaimers]

NIH Support

Supported by NIH grant P20 CA86346