GlyCLICK Biotin enables site-specific chelation of IgG with Deferoxamine (DFO)



GlyCLICK DFO is a kit that contains all reagents needed for site-specific conjugation of IgG using DFO as chelator for metal-tagging or radiolabeling. Spin columns with GlycINATOR (EndoS2) enzyme immobilized on agarose beads deglycosylates IgG from several species and subclasses by hydrolyzing any Fc glycoform after the inner most GlcNAc. The GalT enzyme supplied in solution enables incorporation of the UDP-GalNAz regent to the exposed GlcNAc sugar for subsequent click chemistry incorporating the DFO chelator. GlyCLICK DFO is available in kit formats to facilitate chelation of 250 ug or 2 mg of IgG.

Detailed Information



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Characterization using LC-MS will give a complete and clear picture of mass shift associated with successful conjugation if the antibody is analyzed reduced or fragmented. LC or LC-MS analysis of HC from a reduced sample or scFc fragments generated by FabRICATOR digestion provides distinct elution peaks for conjugated scFc or mass shifts corresponding to the degree of labelling (DOL) following conjugation. On the application page you can see an example of such analysis. Analysis of fluorescently labeled antibodies can also be performed by absorbance measurements and calculate the DOL using the Extinction coefficient for the antibody and the label and the correction factor for the label.

All the steps (deglycosylation, azide activation and click reaction) in GlyCLICK are highly efficient. Normally, all molecules are conjugated when using GlyCLICK, resulting in DOL=2. On the application page we have shown by MS-analysis that all molecules have been conjugated and there is no remaining un-conjugated material.

The GlyCLICK technology can be used to conjugate IgG with Desferrioxamine (DFO) using the GlyCLICK DFO kit. The DFO is a chelating agent for radiolabeling with the radioisotope Zirconium-89 (89Zr) for PET-imaging. The Azide Activation kit can be used to conjugate a chelator of choice if it is alkyne-modified (carrying for example DIBO, DBCO or BCN) to be compatible with the click chemistry.

The final step of GlyCLICK labeling kit is based on a copper free click reaction (SPAAC) and it is a strain promoted reaction between an azide and an alkyne-carrying label. As the name indicates the reaction occurs spontaneously due to the strained bond angles. The use of this click reaction that forms stable conjugates, allows you to freely choose label or payload to combine with your IgG as long as it is alkyne-functionalized. Several alkyne-carrying labels are commercially available with cyclooctyne structures such as DIBO, DBCO or BCN.

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