Håkan Roos

Studies of interactions with weak affinities and low-molecular-weight compounds using surface plasmon resonance technology†

Interactions between the immobilized weak‐affinity monoclonal IgG antibody 39.5, which is specific for the glucose‐α1,4‐glucose motif, and various oligosaccharides were studied with surface plasmon resonance technology. The antibody was immobilized at high levels on the surface of the sensor chip and different concentrations of the analytes were injected at 25 and 40 °C. The 39.5 antibody exhibited specific binding to maltose, tetraglucose and maltotriose, with dissociation constants Kd in the range from 0.07 mM (25 °C) to 1.0 mM (40 °C). Association and dissociation rate constants (ka and kd) were rapid and baseline was obtained almost immediately after the end of each antigen injection. This excluded the need for a regeneration step but also made calculation of the kinetic values impossible. Owing to the weak affinity and the small size of the analytes (<1000 Da), a careful design of control surfaces is demanded to exclude artefactual results. Copyright

Evaluation of calibration-free concentration analysis provided by Biacore™ systems

Surface Plasmon Resonance biosensors measure the interaction between a molecule in solution and its interaction partner attached to a sensor surface. Under certain conditions, the observed binding rate can be used directly to obtain the concentration of the molecule in solution, without the use of any standard. This type of assay is referred to as Calibration Free Concentration Analysis, CFCA. By examining experimental conditions, including immobilization levels and temperature, for a range of analytes, and by using global analysis of several sample dilutions, conditions that gave the most robust results were identified. These conditions provided the concentration values that were on average ∼15% lower than those obtained using other methods. The accuracy of the concentration determined may be related to how the analyte is distributed in the dextran matrix and to its distance from the gold surface, and may thereby depend on the conversion of the SPR signal to mass. A good precision of CFCA, ∼8% (n = 21), was demonstrated when this method was used to efficiently guide purification procedures of Interferon α-2a. In this paper, the theory behind CFCA and the future developments, as well as the application of CFCA for absolute and relative concentration measurements (including the assessment of the potency of a biotherapeutic medicine) are discussed, and new evaluation tools that broaden the range of applications, are introduced.

Hydronamic addressing: A flowcell for improved signal quality in bioanalytical systems

This paper reports a new flow cell concept for micro-fluidic applications, using a hydrodynamic addressing principle to direct flow to different parts of the flow cell [1]. The concept is designed and implemented in the context of SPR biosensor technology for biomolecular interaction analysis, where the ability to distinguish multiple detection spots in the same flow cell provides improved signal quality and time resolution of the analyses.

Hydrodynamic Addressing Flow Cell: Design Considerations and Calculations

This paper presents experimental and CFD (Computational Fluid Dynamic) design optimization of the flow cell for hydrodynamic addressing (HA), a concept that was presented earlier this year [1],[2].