Label-free methods for the measurement and characterization of biomolecular interactions are very popular and widely used, e.g. when conducting binding assays and screenings. Here, on-rates, off-rates, and affinities are determined based on an experimental set-up, in which a ligand is captured to a sensor’s surface; the ligand then interacts with an analyte that the sensor is probed with. Applied assay technologies comprise surface plasmon resonance (SPR), e.g. from BiacoreTM/Cytiva (formerly GE Healthcare), bio-layer interferometry (BLI), e.g. from FortéBio/Sartorius, and switchSENSE® from Dynamic Biosensors.
The stable, uniform and oriented, i.e. site-directed, immobilization of the ligand is of considerable importance for the reliable determination of the binding kinetics of ligand to analyte.
In general, these assays are distinguished by the immobilization of the ligand to the sensor’s surface:
- Direct immobilization assays:
Covalent linking via lysines or cysteines of the ligand and click chemistry
- Indirect immobilization assays or capture kinetics assays:
Capturing the ligand via specific binding molecules like antibodies or Nanobodies in a site-directed, i.e. Fc-domain specific manner or via antibodies or Nanobodies against protein- or peptide-tags like GFP, GST, MBP, Spot-tag, His-tag, etc.
Common capture molecules for antibodies are anti-Fc specific antibodies and Nanobodies like Nano-CaptureLigands™, or protein A, protein G, protein A/G.
Compared to direct immobilization assays, the benefits of capture kinetics assays are
- site-directed immobilization,
- regeneration is a standard procedure and does not depend on the captured molecule, because the ligand is removed after every cycle,
- the ability to capture unpurified monoclonal antibodies from crude samples,
- easier optimization of capture ligands’ density.
ChromoTek’s Nano-CaptureLigands are optimized for the site-directed, specific, dense, and gentle immobilization of antibodies to biosensors. Conveniently, no antibody biotinylation is required. Their high selectivity enables the specific immobilization of antibodies, antibody fragments, and Fc-fusions. Because of their high affinity, they provide a stable baseline with negligible antibody dissociation. Nano-CaptureLigands’ chemical stability allows for a frequent regeneration for multiple reuse and the application of standard protocols for faster protocol development.PRODUCTS
Nanobodies as capture ligands
Nanobodies may be used as capture ligands because of their extraordinary chemical stability and high affinity; this approach may be also referred to as indirect ligand capture or immobilization. In general, a very strong interaction between the capturing molecule, i.e. single domain antibody, and the ligand is needed to avoid ligand dissociation from the sensor surface.
Besides Nano-CaptureLigands, which consist of biotinylated anti-immunoglobulin VHHs, also the biotinylated anti-GFP VHH/ Nanobody is a very valuable tool for capture of GFP-fusion proteins because of its extremely high 1 pM affinity, as determined by switchSENSE® technology. Note, also GST, MBP, and Spot VHHs and other Nanobodies from ChromoTek are very suitable for the immobilization of corresponding fusion proteins in biosensor assays.
Antibodies as capture ligands
Antibodies may also be used as capture ligands since there are many antibodies commercially available. They do, however, need to have high affinities to avoid ligand dissociation from the sensor surface. In addition, immobilization of the antibody to the sensor surface can affect its affinity to the ligand. Both, direct immobilization by NHS chemistry or biotinylation may modify the antibody’s paratope and hence may affect its capture efficiency. Compared to Nanobodies, conventional antibodies are rather fragile. Thus, immobilized IgG may not be regenerated effectively and frequently.PRODUCTS