Biomolecular Interaction Analysis is not limited to proteins. Interactions between DNA - DNA, DNA - protein, lipid - protein and hybrid systems of biomolecules and inorganic surfaces can be investigated.
Interaction studies can be used to observe if two or more interactants bind to each other. It can also be used to measure how strong the interactions are, and even to measure the actual association and dissociation rates. In addition, the binding of two interactants can be used to measure the concentration of one of the interactants after making a calibration curve or by means of the calibration free concentration analysis procedure.
Specificity / screening
When screening for ligand binding partners, the main goal is not to obtain accurate kinetic data. The first aim should be to identify as quickly as possible the candidates that bind the best to a ligand (yes–no binding). Examples are small compound screening and epitope binning. These specificity studies are relatively easy to perform. Essentially, an analyte is injected and after a fixed amount of time the response is measured. Plotting the responses against the sample number will reveal the best binders. A possible pitfall is the bulk response caused by the medium in which the analyte is dissolved. However, with the proper reference measurements, this can be easily avoided.
Epitope binning is used to make groups (bins) of compounds (e.g. antibodies, drugs) that bind (or interfere) to the same epitope on the ligand. For instance, in the sandwich method an antibody is immobilized on the sensor surface and the analyte is bound. A second antibody is used to asses if the two antibodies compete for the same binding site. In the tandem method, the ligand is the target for the antibodies. Antibodies are injected sequentially to look for competition for the ligand binding sites.
Competition experiments are often done when the analyte is too small to measure directly with SPR. In surface competition, a constant concentration of a larger analyte is mixed with a concentration range of a small compound. When the small compound binds to the ligand the overall binding response will diminish with the small compound concentration. In solution competition the analyte is mixed with a competitor for the immobilized ligand, effectively lowering the analyte concentration in solution when it binds.
Equilibrium analysis is used to determine the strength of the binding. Two types of experiments can be done.
The first type involves flowing analyte over the ligand until the signal levels out and the net association is equal to the dissociation. This is done with several analyte concentrations. After plotting the reached response versus the analyte concentration, a line can be fitted which provides the equilibrium dissociation constant KD.
The second type of experiment involves putting the two interactants together in a test tube and incubating them until equilibrium is reached. One of the interactants is held constant and the other varied (the analyte) over a range of concentrations. After equilibration, the concentration of free analyte is determined. With some calculations, the equilibrium dissociation constant can be determined.
Kinetic rate analysis
Kinetic rate analysis is used to investigate the behaviour of the system. The analyte is flowed over the ligand and the association rate is monitored in real time. After a while, buffer replaces the analyte and the dissociation rate of the analyte is monitored. Both the association and dissociation curve can be fitted to one of the chosen models to obtain the kinetic rate constants. Assessing the fit and calculated constants will reveal if the model is correct. In addition, the equilibrium constant can be calculated.
Analyte concentrations are measured using sensor surfaces with very high ligand densities. In this way, the binding rate is limited by the diffusion of the analyte towards the surface. The binding rate is then proportional to the analyte concentration. After establishing a standard curve, unknown samples can be measured quickly and accurately.
An alternative method is the Calibration Free Concentration Analysis (CFCA) (1),(2),(3). This method makes it possible to measure the active concentration of a ligand without a calibration curve. The method makes use of the mass transport limitation (km) calculated from the molecular mass (Mr), which occurs when high-density ligand surfaces are used. By injecting the analyte at two different flow rates (e.g. 10–90 µl min-1), the active analyte concentration can be calculated from the slopes of the curves. Some new SPR machines have this method built into the software.
Structure and function
Various techniques are used to study structure-function relationships. Function is measured in terms of specificity (affinity), rate and equilibrium constants as well as thermo-dynamic properties. While with the majority of SPR experiments the interaction conditions are held constant, varying these conditions (e.g. the temperature) can reveal important thermodynamic properties (4),(5). Microcalorimetry, often the method of choice for thermodynamic analysis, measures all components at equilibrium, including solvent effects and brief intermediate states (6). SPR systems are capable of measuring only the specific ligand-analyte interaction at real-time enabling the researcher to obtain both rate and equilibrium constants at the same time. SPR measurements are insensitive to changes in the number of associated water molecules present in the complex, since these changes do not affect the refractive index on the sensor surface (5).
The MP-SPR set-up with full angular scan measurements at multiple wavelengths enables correct determination of thickness and refractive index of nano layers (biological or inorganic) and nanoparticles. Both parameters can be determined at the baseline, after triggering event and after regeneration. This in turn provides an insight into the conformational changes. In MP-SPR triggers can be pH change, temperature change, dry-to-wet change, light trigger (e.g. UV), electric potential change. These types of measurements are useful not only in the life sciences, but also in research of material properties, such as drug release or loading capacity of biomaterials.
|(1)||Pol, E., R. Karlsson, H. Roos, et al. Biosensor-based characterization of serum antibodies during development of an anti-IgE immunotherapeutic against allergy and asthma. Journal of Molecular Recognition 20: 22-31; (2007). Goto reference|
|(2)||Chavane, N., R. Jacquemart, C. D. Hoemann, et al. At-line quantification of bioactive antibody in bioreactor by surface plasmon resonance using epitope detection. Analytical Biochemistry 378: 158-165; (2008). Goto reference|
|(3)||Pol, E., H. Roos, F. Markey, et al. Evaluation of Calibration-Free Concentration Analysis provided by Biacore™ systems. Analytical Biochemistry (2016). Goto reference|
|(4)||Roos, H., R. Karlsson, H. Nilshans, et al. Thermodynamic analysis of protein interactions with biosensor technology. J.Mol.Recognit. 11: 204-210; (1998). Goto reference|
|(5)||Zeder-Lutz, G., E. Zuber, J. Witz, et al. Thermodynamic analysis of antigen-antibody binding using biosensor measurements at different temperatures. Analytical Biochemistry 246: 123-132; (1997).|
|(6)||Roos, H. and R. Karlsson Finding the route form structure to function: modifying temperature. (1999).|