Antibodies, like other proteins, can be covalently modified in many ways to suit the purpose of a particular assay. Many immunological methods involve the use of labeled antibodies and a variety of reagents have been created to allow labeling of antibodies. Enzymes, biotin, fluorophores and radioactive isotopes are all commonly used to provide a detection signal in biological assays.
Antibody structure and modification sites
Understanding the functional groups available on an antibody is the key to choosing the best method for modification, whether that be for labeling, crosslinking or covalent immobilization. Most antibody labeling strategies use one of three targets:
- Primary amines (–NH2): these occur on lysine residues and the N-terminus of each polypeptide chain. They are numerous and distributed over the entire antibody.
- Sulfhydryl groups (–SH): these occur on cysteine residues and exist as disulfide bonds that stabilize the whole-molecule structure. Hinge-region disulfides can be selectively reduced to make free sulfhydryls available for targeted labeling.
- Carbohydrates (sugars): glycosylation occurs primarily in the Fc region of antibodies (IgG). Component sugars in these polysaccharide moieties that contain cis-diols can be oxidized to create active aldehydes (–CHO) for coupling.
Antibody structure and labeling sites.
Primary amines as antibody labeling sites
The most common target for antibody labeling or conjugation is primary amines, which are found primarily on lysine residues. They are abundant, widely distributed and easily modified because of their reactivity and their location on the surface of the antibody.
Primary amines can be targeted using several kinds of conjugation chemistries. The most specific and efficient reagents are those that use the N-hydroxysuccinimidyl ester (NHS ester) reactive group. Many biotinylation and fluorescent labeling products are commercially available pre-activated with NHS-ester group.
Other amine-targeted strategies are commonly used to conjugate antibodies to enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP); these include glutaraldehyde and reductive amination crosslinking approaches. Thermo Scientific AminoLink Plus Coupling Resin uses reductive amination to covalently immobilized antibodies through primary amines.
In any particular antibody clone, lysines (primary amines) might occur prominently within the antigen binding site. Thus, the lone drawback to this labeling strategy is that it occasionally causes a significant decrease in the antigen-binding activity of the antibody. The decrease may be particularly pronounced when working with monoclonal antibodies or when attempting to add a high density of labels per antibody molecule.
Sulfhydryls as antibody labeling sites
The second useful target for covalently labeling antibodies is sulfhydryls. These groups exist in proteins under reducing conditions but more often are found in native proteins (including antibodies) in oxidized form as disulfide bonds (cystine). Disulfide bonds are important contributors to antibody function as they participate in the tertiary structure of each subunit, covalently connect heavy and light chains, and connect the two antibody halves at the hinge region.
Conjugation at sulfur atoms requires that the thiols exist as free suflhydryls. Thus, to label an antibody, at least some of the native disulfide bonds must be cleaved with reducing agents. Because disulfides in the hinge region are the most susceptible to reduction, it is possible to selectively cleave only these disulfides and thereby to split the antibody into monovalent halves without damaging the remaining structure and antigen-binding sites.
When it can be accomplished, labeling antibodies at hinge-region sulfhydryls ensures consistent labeling at a defined location. In contrast with amines-targeted labeling, this provides greater certainty that antigen binding sites will not be inactivated and that the population of antibody molecules in a sample will acquire the same density of label.
Reagents that are activated with maleimide or iodoacetyl groups are the most effective for sulfhydryl-directed conjugation. Many biotin, fluorescent and enzyme labeling reagents are available pre-activated with maleimide groups. Thermo Scientific SulfoLink Coupling Resin uses iodoacetyl chemistry to immobilize antibodies through sulfhydryl groups.
Carbohydrates as antibody labeling sites
The third useful target for labeling antibodies is carbohydrate moieties. Because glycosylation sites in antibodies are predominantly found on the Fc portion of the antibody, they can often be modified without significantly affecting the antigen-binding capacity.
Labeling carbohydrates requires more steps than labeling amines because the carbohydrates must first be oxidized to create reactive aldehydes; however, the strategy generally results in antibody conjugates with high activity.
Aldehyde-activated (oxidized) sugars can be reacted directly to primary amines through reductive amination (mentioned above) or to reagents that have been activated with hydrazide groups. Several hydrazide-activated biotinylation reagents are available. GlycoLink Coupling Resin uses reductive amination to immobilize antibodies through carbohydrate groups that have been oxidized. This antibody immobilization method can even be used to for immunoprecipitation.
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