Genovis » GlyCLICK DFO

Description

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

Application	Antibody Conjugation, In vivo imaging (PET)
Product Group	GlyCLICK
Source/Origin	Cloned from S. pyogenes, Produced in E. coli (His-tagged), Engineered b-1,4-galactosyltransferase (Y289L)
Product Composition	Enzymatic components: Immobilized GlycINATOR (EndoS2) on agarose beads (20% EtOH), beta-1,4-galactosyltransferase (GalT)
Specificity	GlycINATOR hydrolyzes Beta-1,4-linked GlcNAc residues on all IgG Fc-glycoforms for site-specific azide activation using GalT and complete labeling using SPAAC
Digestion Site	IgG Fc N-glycans
Substrates	Cow IgG, Fc-fusion proteins, Goat IgG, Horse IgG, Human IgG1, Human IgG2, Human IgG3, Human IgG4, Monkey IgG, Mouse IgG, Rabbit IgG, Rat IgG, Sheep IgG
Reaction Conditions	Deglycosylation (GlycINATOR): 30 min incubation at 37°C, Azide Activation (GalT): O/N incubation at 30°C, Labeling (SPAAC): O/N incubation at 25°C, *The protocol can vary depending on kit size/amount of IgG
Digestion Buffers	TBS (pH 7.4)
Buffer Compatibility	GlyCLICK steps are performed in TBS buffer, provided in the kit., For purification of azide activated Ab, TBS or PBS can be used., Sodium azide interferes with the click reaction and must be avoided., Avoid phophate buffers, not compatible with buffer additive (precipitation).
Additives/Co-factors	GalT requires buffer additive for activity., Buffer additive is included in the GlyCLICK kit.
Inhibitors	Denatured IgG
Unit Definition	Components for antibody conjugation of up to 0.25-2 mg IgG
Label	DFO

FAQs

How do I perform quality control of the conjugation process?

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.

What is the conjugation efficacy using GlyCLICK?

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.

Which isotopes can I use to radiolabel IgG after chelation using GlyCLICK?

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.

Why do I need to use an alkyne-functionalized label with GlyCLICK?

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.