Antibody labeling chemistries

Covalent conjugation via NHS ester groups is the most common antibody labeling method, which involves attaching the label of interest to the amine groups of lysine residues. The NHS ester method uses fluorescent dyes with a reactive N-Hydroxysuccinimide (NHS) group (Fig. 3). After reacting with the antibody, any surplus reactive dye is removed through various separation processes. These separation processes usually take advantage of the label’s small size compared to the antibody conjugate, as in gravity column chromatography. While these separation steps can result in conjugate losses, it allows removing of unbound dye, which can cause high background in your experiment.

The two-tag method is generally used to conjugate protein molecules, for example, HRP. This is because both the antibody (Ab) and label (L) have multiple lysine groups, and there is a risk of forming Ab-Ab or L-L homodimers or high-molecular-weight aggregates.

The main advantage of the two-tag approach is that it provides a controlled reaction to form conjugated antibodies. However, pre-conjugation separation steps can lead to the loss of valuable antibody reagent (and label), uncertainty over the amount of recovered antibody, batch-to-batch variation, and lack of scalability.

All labeling methods described above rely on specific aminoacidic residues in the chains of the antibody. However, the number and location of these amino acids can vary depending on the antibody chain sequences, and certain amino acids may belong to the antigen-binding site. Labels binding near the paratope can interfere with the antibody structure, charge, and affinity, and therefore its function.

Site-specific conjugation ensures that labeling happens far from the antigen-binding site, so labels do not create steric hindrance that could impact the antibody’ binding affinity and avidity. Through enzymatic activation or modification of specific domains in the F_C region or antibody sequence engineering, it is possible to target specifically modified amino acids at pre-defined locations of the antibody chain to achieve a very high level of control and reproducibility over antibody labeling.

An alternative to primary amine modification is to target sulfhydryl groups, also called thiols. Pairs of cysteine sulfhydryl groups exist in proteins and antibodies as intra-chain disulfide bonds (–S–S–)and therefore require chemical modification (i.e., reduction) to become available in their reactive form (-SH) for antibody labeling. Upon each disulfide bond reduction, two thiol groups are formed, which can be paired with thiol-reactive compounds, such as maleimides and pyridyl disulfides.

Targeting cysteine residues allows us to attach the label of interest away from the binding site region. However, not all antibodies will tolerate the chemical reduction, leading to the creation of unstable half antibodies that show lower avidity due to the presence of only one binding site.

Glycans are carbohydrate-based polymers made by all living organisms. This site-specific method targets carbohydrate residues on the Fc region of all IgG antibodies. Hence, this method allows us to attach the desired label far away from the antigen binding site.

The original chemical method was relatively complex and harsh, depending upon cis-diol oxidation to create aldehydes groups that react with hydrazides. Innovative and gentler approaches involve using enzymes to specifically attach an azido moiety to the N-linked glycans on the heavy chains of an IgG antibody; once azido–modified, the label of interest can be attached to the antibody via “click” chemistry.