Deferoxamine B (DFO) is the most widely used chelating agent. CD BioGlyco provides clients with homogeneous and well-defined DFO-antibody conjugates through glycan remodeling.
In medical and biological applications, specific tags are often added to antibodies for analysis and detection, such as immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), or fluorescence-activated cell sorting (FACS). Traditional conjugation methods are non-specific and it is difficult to control the labeling position and exact amount of each antibody. And non-specific labels may cover the active site of the antibody, thereby affecting the ability of the antibody to interact with its antigen. Various methods have been developed for conjugating labels to specific sites on antibodies. These site-specific methods rely on engineering the antibody itself before conjugation, such as chemoenzymatic techniques and chemical approaches. The chemo-enzymatic method achieves site-specific conjugation by modifying glutamine residues mediated by transglutaminase (TGase) or glycan remodeling of asparagine N-glycans in the conserved Fc region. Chemical conjugation is achieved by the selective reduction of disulfides followed by alkylation or modification of the N-terminal amino group.
Fig.1 Antibody conjugates constructed via a convergent assembly. (Sadiki, et al., 2020)
Chemoenzymatic methods are usually carried out under mild reaction conditions. More than 60 glutamine (Gln) residues are present in antibodies, but only one conserved residue is modified in heavy and light chains. After deglycosylation of the two asparagine amides at the C-terminus of this conserved residue, the corresponding amine substrates are efficiently modified under the catalysis of TGase. Since almost all IgG-type antibodies have only one glycan at a conserved site in the Fc region. Therefore, it is also possible to reconstruct glycans for site-specific labeling by adding or removing sugars at these conserved sites and then installing an unnatural sugar containing a handle.
DFO is a hexadentate siderophore containing three hydroxamate groups for chelating metals and a primary amine tail for binding to biomolecules. As a chelating agent for several metal ions such as zirconium, it shows good stability. At CD BioGlyco, we have developed advanced Site-Specific Antibody Conjugation technology. We attach DFO to the specific position of the antibody (from humans, mice, rats, goats, sheep, monkeys, rabbits, cows, and horses) through glycan remodeling. Our specific method is as follows:
1) Trim the antibody's native glycan by β-galactosidase or hydrolyze the Fc glycan to the innermost N-acetylglucosamine (GlcNAc) by endoglycosidase.
2) Install a functionalized unnatural sugar containing a handle to activate the antibody.
3) Under specific conditions, the DFO is conjugated to an activated antibody containing a handle.
Fig.2 Process of site-specific antibody conjugation with DFO. (CD BioGlyco)
CD BioGlyco is a leading service provider specializing in Antibody Glycoengineering. We provide clients with high-quality site-specific antibody conjugation services. If you are interested in our services, please contact us for more details.
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