In this post, we’ll focus on the tools Alces Bioworks uses to identify and characterize Alcein™ affinity reagents in the lab. Knowing the affinity of a protein-protein interaction could be useful for many applications: screening for tight-binding therapeutics in drug discovery, evaluating an antibody for analytical assays, or studying an interaction as part of basic research. But imagine that you asked Alces Bioworks to create an Alcein™ that binds your target of interest. How do we measure the strength of this binding interaction?
First, let’s look briefly at how affinity is quantified. In this context, affinity is a measure of how tightly a protein binds its ligand. Here, it would be a measure of how tightly an Alcein™ binds its target protein. Imagine an Alcein™ ‘A’ binding to a target protein ‘T’ to form the complex ‘AT.’ The chemical equilibrium can be described by this simple equation:
A + T ⇌ AT
This equilibrium is the result of the complex AT forming, which happens at a velocity that is a function of the ‘on rate,’ or association rate constant:
A + T ⇀ AT
and also the complex AT dissociating at a rate called the ‘off rate,’ or dissociation rate constant:
AT ⇀ A + T
The dissociation equilibrium constant (Kd), can be calculated by dividing the off-rate (koff, expressed in s-1) by the association rate constant (kon, expressed as M-1s-1). This is the ratio that tells you the strength of the interaction.
Kd = koff / kon
A dimensional analysis shows that Kd is expressed as molarity (M) or using smaller units such as µM, nM, or even pM. Smaller values indicate tighter interactions.
Numerous techniques for determining binding affinities have been used throughout the years, including Kinetic Exclusion Assays (KinExA), Nuclear Magnetic Resonance (NMR), Fluorescence Anisotropy (FA), and Isothermal Calorimetry (ITC). However, for most biopharmaceutical applications, the two techniques that dominate the field due to their ease of use and throughput are Surface Plasmon Resonance (SPR) and Biolayer Interferometry (BLI). SPR has long been considered the gold standard for protein affinity determination. Biacore, owned by Cytiva, introduced the first commercial SPR instrument in 1990. Today, several companies make SPR instruments, for example, Bruker, Carterra, and Nicoya. BLI was introduced commercially in 2005 by ForteBio, now owned by Sartorius, which produces the Octet instrument series. Gator Bio also manufactures BLI-based devices. This technology has become a respected alternative to SPR.
Both SPR and BLI instruments monitor proteins (e.g., an Alcein™ A) binding to a sensor surface covered with a ligand (e.g., the target protein T) and plot the interaction in real time. A major advantage of both methods is that they are label-free, meaning that you don’t have to attach fluorescent or radioactive compounds to your proteins of interest. However, one of the two interacting proteins must be immobilized on the sensor. Since BLI and SPR rely on different physical phenomena, the two techniques have different strengths.
SPR detects differences in the refractive index near a sensor surface when analytes bind to immobilized ligands. The SPR signal occurs when polarized light is reflected off a thin metal film. The metal has oscillating free electrons (or surface plasmons), which influence the intensity of the reflected light. When the protein of interest (e.g., an Alcein™) binds to the target protein immobilized on the sensor surface, it causes a change in the local refractive index, which shifts the resonance angle of the reflected light. The change can be detected and plotted as the association and dissociation curves.
You can conveniently watch as the binding curve rises when the Alcein™ comes in contact with the surface and attached target protein, and then falls as it is “washed off” in the dissociation buffer. The end result is the characteristic plot like the one below (Image adapted from Cytiva Application Guide: Kinetics and affinity measurements with Biacore systems).
While the dissociation rate is concentration-independent, the association rate (seen here as the “slope” of the curve starting at time 0) is dependent on the protein analyte concentration (binding curves of different colors correspond to different analyte concentrations). To get an accurate fit of your data, you will need to determine full binding saturation (or Rmax,) which is best estimated by high concentration samples (like the red curve), as well as determine the binding rate of a few concentrations in the “sweet spot” (like the brown to the green curves). With live monitoring of the binding data, a moderately experienced scientist will be able to gauge immediately if they need to adjust the surface ligand or solution analyte concentrations.
In contrast, BLI does not rely on surface plasmons. It detects changes in the interference pattern of light channeled in a biosensor made of optical fiber. A bio-compatible matrix coats the tip of the sensor, and proteins binding to this matrix trigger a signal by modulating the light traveling down the optical fiber.
The BLI signal is based on the principle of light interference, which occurs when some of the light traveling down the optical fiber is reflected internally off the internal surface of the sensor tip. (See figure taken from Jug et al. 2024.) The biomolecular layer on the outside of the sensor surface forms a second surface upon which some of the light can also be reflected. The thickness of the biomolecular layer influences the interference of the two reflected beams of light. As the target protein-Alcein™ complex forms or dissociates on the sensor tip, the thickness of the layer changes, resulting in a shift in the interference spectrum. The change can be monitored and plotted as the association and dissociation curves.
The differences between BLI and SPR detection techniques have some implications. BLI uses a multi-well plate format (e.g., 96-well) with multiple sensors (fiber optics), allowing for the parallel analysis of many samples at once. This “dip and read” approach is significantly faster for large screening campaigns, although high-throughput SPR devices are also available. The absence of a microfluidics system means there are no clogging issues, making BLI more tolerant of crude or dirty samples like cell lysates or hybridoma supernatants. This also makes the system easier to maintain. Both the equipment and consumables (biosensors) for BLI are less expensive than for SPR, making it a more accessible option for labs with a limited budget.
SPR is considered the gold standard for detailed kinetic analysis, offering superior resolution and the ability to measure interactions with small molecules (down to ~100 Da). The continuous flow system in SPR allows for precise, repeatable measurements of association rates and can even provide thermodynamic parameters in some cases. With a longer history, SPR is the established technique for regulatory submissions (e.g., FDA), making it standard practice for drug development and validation.
For both systems, sensors that rely on the capture of proteins fused to affinity tags (e.g., His-tag or FLAG-tag) are available and facilitate immobilization of proteins or their detection via a secondary reagent. NHS-amine chemistry is also available and commonly used to attach proteins to sensors without the need for affinity tags.
To sum up, both SPR and BLI offer direct measurement of protein-protein binding interactions implemented in reliable instruments, and both are extensively documented in the scientific literature. Alces Bioworks has used both methods to determine the affinity of custom-generated AlceinsTM to target proteins of interest. Due to its lower cost, ease of use, and higher throughput, BLI has become a valuable tool for us to rapidly screen protein-protein interactions. We then use SPR to validate and characterize the binding affinities of the most promising Alceins™ we identified in the screen. Look out for upcoming blogs detailing our case studies.