The SiteClick™ antibody labeling system

(See a list of the products featured in this article.)

The SiteClick™ system represents a new paradigm for the universal site-selective labeling of antibodies. This modular, click chemistry–mediated method allows you to enzymatically label essentially any antibody on its heavy chain N-linked glycans. In contrast to standard antibody labeling techniques, which can be tedious and inconsistent, the SiteClick™ site-selective approach produces highly robust and reproducible labeling of antibodies with an impressive choice of detection molecules.

Advantages of SiteClick™ labeling

The site-selective labeling provided by the SiteClick™ system (Figure 1) prevents disruption of the antigen-binding domain that can occur with traditional amine- or thiol-reactive labeling reagents and eliminates the need to genetically engineer labeling sites into the antibody prior to modification. This site-selective strategy is especially important when labeling monoclonal antibodies that contain lysine residues in or around the antigen-binding domain, as labeling of these sites can disrupt antigen binding. Monoclonal antibodies can also have variable sensitivities to disulfide bond–reducing agents used in reductive cysteine labeling, leaving partially dissociated antibody fragments that result in decreased antibody binding and yield.

The new SiteClick™ system allows simple and gentle site-selective attachment of detection molecules to heavy chain N-linked glycans—far from the antigen-binding domain—providing excellent reproducibility from labeling to labeling and from antibody to antibody. A number of different detection molecules can be site-selectively attached to the heavy chain glycans—including phycobiliproteins (e.g., R-PE), Qdot® probes, fluorescent dyes, metal-chelating compounds, and other small molecules like biotin—allowing multiplex analysis with antibodies from the same species (Figure 2).

SiteClick antibody labeling systemFigure 1. The SiteClick™ antibody labeling system. The first step in the SiteClick™ antibody labeling process involves removal of terminal galactose residues from the heavy chain N-linked glycans using β-galactosidase, exposing essentially all possible modifiable GlcNAc residues. Second, the free terminal GlcNAc residues are activated with azide tags by enzymatic attachment of GalNAz to the terminal GlcNAc residues using the GalT(Y289L) enzyme. In the third step, the azide residues are reacted with the dibenzocyclooctyne (DIBO)-functionalized probe of choice (e.g., Alexa Fluor® 488 DIBO alkyne). The average degree of labeling is 3–3.5 labels per antibody.

Immunocytochemistry with SiteClick™ labeled antibodies  
Figure 2. Immunocytochemistry with SiteClick™ labeled antibodies. HeLa cells were fixed, permeabilized, and incubated with 10 nM Qdot® 655 anti–golgin 97 antibody (magenta). This conjugate was generated using the SiteClick™ Qdot® 655 Antibody Labeling Kit and mouse monoclonal anti–golgin-97 antibody (clone CDF3). After antibody incubation, the cells were counterstained with NucBlue® Live (blue) and ActinGreen™ 488 (green) ReadyProbes™ reagents prior to imaging.

Site-selective labeling of antibody glycans

In general, IgG antibodies contain two N-linked glycans attached to specific conserved asparagine residues located in the antibody heavy chain Fc domain. These sugar chains, predominantly complex biantennary glycans with two terminal branches, are structurally quite homogeneous, and the terminal sequences of the glycan branches are highly consistent [1]. Most of the antibody glycan branches terminate with galactose-N-acetylglucosamine (Gal-GlcNAc-) or with N-acetylglucosamine (GlcNAc-). Removal of the terminal Gal residue with β-galactosidase unmasks the majority of terminal GlcNAc labeling sites for the subsequent enzymatic β-galactosyl transferase (GalT) reaction (Figure 1). Although a small percentage of antibodies also contain terminal sialic acids, our experiments have shown that the additional removal of terminal sialic acid residues produces little difference in the final degree of antibody labeling.

After cleavage of terminal Gal residues by β-galactosidase, each N-linked glycan will contain, on average, 2 terminal GlcNAc residues per heavy chain (4 terminal GlcNAc per antibody). In practice, the degree of labeling of mouse and rabbit monoclonal antibodies, as determined by subsequent attachment of fluorescent DIBO dyes, averages between 3 and 4 labels per antibody. The SiteClick™ method is compatible with antibodies from a number of different species including, but not limited to, human, rabbit, mouse, rat, goat, hamster, and chicken. Additionally, SiteClick™ labeling is effective with several antibody classes such as IgG, IgM, and IgY; note that chicken IgY antibodies have 6 heavy chain glycans instead of 2 and therefore can be labeled to a higher extent.

GalT(Y289L) enzymatic labeling coupled with a click reaction

The SiteClick™ enzymatic labeling approach takes advantage of the modified β-GalT1 enzyme GalT(Y289L), which is substrate permissive and attaches azide-modified N-acetylgalactosamine (GalNAz) selectively to terminal GlcNAc residues on N-linked antibody glycans [2,3] (Figure 3). These azide-activated antibodies are then reacted with dibenzocyclooctyne (DIBO)-functionalized molecules in a copper-free click reaction, which produces a strong and stable bond between the DIBO detection molecule and the azide-modified antibody [4]. By coupling GalT(Y289L) enzymatic labeling with a click reaction, the SiteClick™ system achieves highly efficient and reproducible site-selective labeling of antibodies, regardless of their originating species or antibody class.

Site selectivity of SiteClick™ antibody labeling  
Figure 3. Site selectivity of SiteClick™ antibody labeling. (A) A β-tubulin monoclonal antibody was azide-activated using the GalT(Y289L) enzyme, labeled with DIBO-functionalized Alexa Fluor® 488 dye, and then analyzed by gel electrophoresis (left panel). Once imaged, the same gel was post-stained with SYPRO® Ruby Total Protein Gel Stain (right panel), showing that only the heavy chain of the antibody is click-labeled with the Alexa Fluor® 488 DIBO alkyne. (B) Anti–β-tubulin mouse monoclonal antibodies were labeled with a small-molecule DIBO-PET chelating agent (L) or left unlabeled (U) (left two lanes) and then post-treated with PNGase F, which selectively cleaves N-linked glycans from asparagine residues (right two lanes). After treatment with PNGase F, both chelator-labeled (L) and unlabeled (U) species are shifted to the same lower molecular weight, confirming the site-selective labeling of heavy chain N-linked glycans.

New SiteClick™ R-PE and Qdot® probe antibody labeling kits

Our newest SiteClick™ products include R-PE antibody labeling kits, which are designed especially for flow cytometry applications, as well as several Qdot® antibody labeling kits that produce conjugates for imaging or flow cytometry applications. These kits include the key reagents for site-selectively labeling 100 µg of purified antibody with either the R-PE or Qdot® fluorophores. The kits are configured to provide an easy-to-follow workflow that will allow novice and experienced scientists alike to obtain efficient antibody labeling every time (Figure 4). And, because antibody glycans are highly conserved, even between different species, the reproducibility of SiteClick™ labeling for different antibodies is very high, precluding the need to optimize labeling of each newly acquired antibody.

Efficient antibody labeling with SiteClick system  
Figure 4. Efficient antibody labeling for both novice and experienced scientists.
The SiteClick™ Qdot® 605 and SiteClick™ R-PE Antibody Labeling Kits were used by both a novice user and an expert user to label an anti-CD4 monoclonal antibody in triplicate. The labeled antibodies were used to label CD4-positive cells isolated from a single human blood donor, with subsequent analysis by flow cytometry; the reference sample is a commercially available R-PE anti-CD4 antibody conjugate. The data show percentages of CD4-positive cells relative to total cells, and the error bars indicate variation in the triplicate kit labelings for each user. The novice user had never before performed protein bioconjugations, yet obtained labeling efficiencies equivalent to those obtained by an expert user.

Custom and OEM site-selective antibody labeling services

  • Do you have a “difficult-to-label” antibody?
  • Does your antibody lose activity after labeling?
  • Are you looking for a service that would custom-label your antibody in a site-selective manner?

Our Custom Services department offers complete custom antibody labeling services, with many detection options that include Alexa Fluor® dyes, Qdot® probes, R-PE, biotin, and others. If you are interested in the GalT(Y289L) enzymatic antibody labeling kit alone (without the click reaction components), or in the custom synthesis of DIBO-modified reagents, please contact our Custom Services representatives for a quote.

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