Antibody Labeling Chemistries
August 25th, 2017
Expedeon – easy to use antibody, protein and peptide labeling technologies
Anyone new to this field will find a bewildering number of antibody labeling protocols in the literature. It is difficult for the newcomer to know what the critical parameters are and what procedure should be applied in any given situation. With traditional labeling methods, a basic understanding of the principles of chemical modification is required because the antibody and/or label must be chemically ‘activated’ before the labeled antibody (or ‘conjugate’) can be formed.
This guide is a user friendly tool that allows you to learn the basics of commonly targeted groups for labeling. It also describes Expedeon’s antibody labeling technologies, which massively simplify the production of labeled antibodies.
Antibody Labeling Approaches and Sites
Many assay formats and other antibody related applications require antibodies to be labeled. Modification is carried out by covalent attachment of antibody to label. Labels may include enzymes, fluorophores, reactive molecules such as biotin and streptavidin and other haptens. ELISA assays and flow cytometry are two common applications which rely on labeled antibodies, the former usually using antibodies labeled with horseradish peroxidase (HRP) or alkaline phosphatase (AP) and the latter using fluorescently labeled antibodies.
There are three principle approaches that can be used to label antibodies, and indeed any other protein molecule. These are via the following reactive groups:-
- Amines (-NH2)
- Sulfhydryl (-SH)
Diagrammatic representation of sites available for conjugating antibodies, or antibody fragments to labels. Labels can be targeted at Fab or Fc regions by modifying amine or sulfhydryl groups, the antibody shown being labeled at a primary amine (indicated by yellow star and L) in the Fc region. Carbohydrate groups occur mainly in the Fc region.
The most common targets for modifying protein molecules are primary amine groups that are present as lysine side chain epsilon-amines and N-terminal ?-amines. Amine groups are distributed throughout the antibody and are easily modified due to their steric accessibility. Labeling methodology involves reacting primary amines with either NHS and sulfo-NHS esters, forming an amide bond.As the amine groups are distributed throughout the antibody the resulting conjugate may have the label distributed randomly across all the antibody domains.
A disadvantage to using amine groups to attach label to antibody is that some monoclonal antibodies may have amine groups within the antigen binding site, meaning that the labeling process may reduce the affinity of the antibody for its antigen. In the great majority of applications this is rarely a problem when using Lightning-Link® kits. Another possible issue is that antibodies are frequently found in solution with other amine-bearing molecules such as BSA, which will also react with the label, effectively blocking the reaction. Similarly buffers containing amines (such as tris and glycine) will compete with the reaction. Expedeon offers simple solutions to overcome these issues to facilitate antibody labeling.
Expedeon’s Lightning-Link®, InnovaCoat® and Thunder-Link® ranges offer rapid technologies for conjugating antibodies at amine groups. Periodate activated HRP is also available for NH2 labeling of any amine containing molecules with horseradish peroxidase.
An alternative to primary amine modification is to label sulfhydryl groups. However, antibodies do not have free sulfhydryl groups available, and therefore antibodies require chemical modification such as reduction to make such groups available. Sulfhydryls are present in antibodies in their oxidised form as disulfide bridges, where they contribute to the tertiary structure of the antibody, both joining heavy and light chains and attaching the two halves at the hinge region, and also as intra-chain disulphide bonds.
Sulfhydryl-reactive groups on labels include maleimides, iodoacetyls and pyridyl disulfides. These require free sulfhydryl groups on the antibody for conjugation, so disulfide bonds must first be reduced to create sulfhydryl groups for labeling, or otherwise introduced chemically.
Expedeon offers a range of maleimide activated labels and InnovaCoat® GOLD – Maleimide 20OD kits for labeling with sulfhydryls. Through using Fab’ antibody fragments and attachment via the sulfydryl groups – the active domain of the antibody fragment can be orientated in such a way that is not inhibited by the labeling process (see diagram below).
Carbohydrate residues appear primarily in the Fc region of antibodies. Targeting them enables antibodies to be labeled at a site remote from the antigen binding site.
In order to use them to conjugate labels, carbohydrates containing cis-diols need to be oxidized to create aldehydes (–CHO). This requires a relatively complex protocol using periodic acid. The aldehydes can then be reacted with hydrazide activated labels.
One drawback of using carbohydrate groups to label antibodies is that some monoclonals are not glycosylated, and thus cannot be conjugated this way.
Expedeon simple antibody labeling kits – random, site specific, and orientated.
As outlined throughout the text there are a variety of sites which can be targeted on an antibody for labeling. Expedeon’s easy to use antibody labeling technologies utilize these various approaches to provide amine, sulfhydryl or carbohydrate based conjugations, including site specific and orientated approaches – which are highlighted in the tables above; to learn more about the technologies please click the relevant links: