SmartEnzymes GlyCLICK workflow

Site-specific Conjugation of IgG

GlyCLICK is a three-step conjugation technology for site-specific and quantitative conjugation of IgG from several species and subclasses.

Specific Fc N-glycan hydrolysis allows for site-specific conjugation at the core GlcNAcs using robust click-chemistry and results in a degree of label (DOL) or drug-antibody ratio (DAR) of 2. The reliable performance ensures quantitative conjugation and intact immunoreactivity for sensitive applications.

The technology is available in a range of kit formats, including conjugation with fluorophores, biotin, DFO, or toxins for ADC development. The format "Azide Activation" renders the antibody azide-activated, for conjugation with any alkyne-reactive label of choice.
 

  • Site-specific conjugation of IgG
  • Reliable performance ensuring quantitative conjugation and preserved immunoreactivity for sensitive applications
  • Stable and homogenous conjugation generates a degree of labeling (DOL) of 2
Human IgG1-4, IgG from mouse, rabbit, rat, monkey, sheep, goat, cow and horse
Conjugation occurs at the Fc N-glycan sites
2-3 day protocol
Fluorophore, Biotin, DFO, MMAE, PNU, Azide Activation

GlyCLICK® Fluorophore

Site-specific conjugation of IgG with Alexa Fluor® 488, 555 or 647

GlyCLICK® Biotin

Site-specific conjugation of IgG with biotin

GlyCLICK® DFO

Site-specific conjugation of IgG with DFO

GlyCLICK® ADC

Site-specific conjugation of IgG with MMAE or PNU

GlyCLICK® Azide Activation

Site-specific conjugation of IgG with azide-alkyne click chemistry

About GlyCLICK®

GlyCLICK workflow

How GlyCLICK works

  1. Deglycosylation
    The Fc specific endoglycosidase GlycINATOR® (EndoS2)1 hydrolyzes the Fc glycans to the inner most GlcNAc moiety on several subclasses and species of IgG. The enzyme removes all glycoforms, including; high-mannose, hybrid, complex, and bisecting type glycans.
  2. Azide Activation
    Azide-containing UDP-GalNAz is enzymatically attached to the exposed GlcNAc using the ß-1,4-Galactosyltransferase Y289L* (GalT) to generate an azide-activated antibody that is reactive with an alkyne-carrying label.
  3. CLICK Reaction
    The azide activated antibody is conjugated with a functionalized label2 of choice in a biorthogonal reaction through strain-promoted copper-free click chemistry (SPAAC)3 to form a stable triazole.

Applications

FAQ and Support

Popular FAQ

GlyCLICK is available in kit formats with AlexaFluor®488, AlexaFluor®555, AlexaFluor®647 or in the Azide Activation kit format for conjugation of a label of choice. With the fluorescent label being alkyne-modified (carrying for example DIBO, DBCO or BCN) it is possible to combine it with the GlyCLICK azide activation kit.

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.

Problems can arise from freezing of the conjugates. We cannot guarantee the quality after freezing and therefore recommend storage at +4°C.

The GlyCLICK 250 ug kit is optimized for 250 ug of IgG as a starting material. We do not recommend using less than 100 ug of starting material in a minimum of 100µL to achieve proper end-over-end mixing on the GlycINATOR columns. Please contact support@genovis.com for assistance if you want to process smaller amounts then 100µg.

The stability of azide activated material varies depending on the antibody and if stabilizing agents have been added. In our labs we have stored azide activated antibodies in TBS buffer without any additives at +4°C for over two years. The azide activation was still OK, and the click reaction generated antibodies with DOL=2. If azide is added as a preservative during long-term storage, it must be removed prior to click reaction.

 

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