Characterizing high affinity interactions between antibodies and their binding partners can bring up technical challenges. Usually, the antibody is used in a low concentration, so in order to measure these interactions, the antibody needs to be labeled with high efficiency. It’s also essential that the label doesn’t affect protein stability or interfere with the binding partner interactions.
To meet these requirements, a site-specific antibody labeling method that is highly efficient, reproducible, and compatible with a broad range of downstream affinity experiments was developed. This method can be used with MST to reliably measure interactions between antibodies and different types of binding partners, including Fc receptors and antigens.
Taking a cue from antibody-drug conjugates does the trick
Standard labeling strategies that involve labeling lysines or carbohydrates don’t work well with antibodies. Since they contain high levels of lysine, there’s a risk of producing heterogeneously-labeled samples or even worse, the binding between antibodies and Fc receptors could be disrupted. Since lysine residues can be such troublemakers, this labeling method focuses on interchain cysteine residues instead. Afterall, that’s how antibody-drug conjugates are produced.
In this application note, the new labeling approach was found to be specific and efficient, and didn’t significantly compromise the structural integrity of Herceptin (Trastuzumab) and Humira (Adalimumab) Furthermore they bound their known Fc receptor and antigen binding partners with low nM affinities in MST binding assays, which was in agreement with previously published Kd values.