Surface plasmon resonance (SPR) measures the binding and dissociation of molecules in real time without fluorescent or radioactive labels. It is the gold standard for determining binding affinity (KD), association rates (ka), and dissociation rates (kd).

Principle

SPR detects changes in the refractive index at a gold sensor surface. One interactant (the ligand) is immobilized on the sensor surface. A solution containing the other interactant (the analyte) flows over the surface. As analyte molecules bind to the ligand, the mass at the surface increases, changing the refractive index. This change is measured continuously and plotted as a sensorgram (response in resonance units vs. time).

Sensorgram

A typical SPR experiment consists of:

1. Baseline: buffer flows over the surface.
2. Association: analyte is injected; the response increases as binding occurs.
3. Equilibrium: the binding rate equals the dissociation rate; the response plateaus.
4. Dissociation: buffer replaces the analyte; the response decreases as the complex dissociates.

Multiple analyte concentrations are injected over the same ligand surface to generate a series of sensorgrams. Global fitting of the data to a 1:1 binding model yields ka, kd, and calculated KD (= kd/ka).

Instrumentation

The Biacore (Cytiva) is the most widely used SPR platform. The sensor chip has a gold surface coated with a carboxymethylated dextran matrix that provides a hydrophilic environment for immobilization. Other platforms include Sierra Sensors, Reichert, and Nicoya.

Immobilization Methods

• Amine coupling: the most common method. The carboxyl groups on the dextran surface are activated with EDC/NHS to form reactive esters that react with primary amines on the ligand. Simple but random orientation.
• Capture coupling: a capture molecule (anti-His antibody, streptavidin, Protein A) is amine-coupled, and the ligand is captured from a crude or purified preparation. This ensures uniform orientation and allows surface regeneration without damaging the ligand.
• Biotin–streptavidin: biotinylated ligands are captured on a streptavidin-coated surface. Near-covalent stability with oriented immobilization.

Applications

• Affinity ranking: screening antibody clones for the highest affinity.
• Epitope binning: determining whether two antibodies bind overlapping epitopes.
• Concentration analysis: measuring active concentration using a calibration-free approach.
• Small molecule analysis: modern SPR instruments detect molecules as small as 100 Da.
• Kinetic characterization: full ka, kd, and KD determination for drug candidates.

Limitations

SPR requires relatively pure ligand and knowledge of the binding stoichiometry. Mass transport limitation (the analyte binds faster than it can diffuse to the surface) can distort kinetics and is addressed by using high flow rates and low ligand densities. Non-specific binding to the surface or matrix must be minimized by optimizing the running buffer (adding surfactant, BSA, or glycerol).