Instruments

The next publications illustrate the use of different SPR-instruments and will discuss the strengths and weaknesses for the different applications.

Papalia, G. A. et al. - High-resolution characterization of antibody fragment/antigen interactions using Biacore T100. Analytical Biochemistry 359: 112-119; (2006). Goto reference

A Biacore T100 optical biosensor was used to characterize the binding kinetics of a panel of antigen binding fragments (Fabs) directed against the PcrV protein from Pseudomonas aeruginosa. We demonstrate that the biosensor response data for each Fab collected from three different surface densities of the antigen could be fit globally to a simple 1:1 interaction model. To further characterize the Fab interactions, binding data were automatically acquired at different temperatures and under different buffer conditions.

Rich, R. L. et al - Extracting kinetic rate constants from surface plasmon resonance array systems. Analytical Biochemistry 373: 112-120; (2008). Goto reference

Surface plasmon resonance imaging systems, such as Flexchip from Biacore, are capable of monitoring hundreds of reaction spots simultaneously within a single flow cell. Interpreting the binding kinetics in a large-format flow cell presents a number of potential challenges, including accounting for mass transport effects and spot-to-spot sample depletion. We employed a combination of computer simulations and experimentation to characterize these effects across the spotted array and established that a simple two-compartment model may be used to accurately extract intrinsic rate constants from the array under mass transport-limited conditions. Using antibody systems, we demonstrate that the spot-to-spot variability in the binding kinetics was <9%. We also illustrate the advantage of globally fitting binding data from multiple spots within an array for a system that is mass transport limited.

Rich, R. L. et al - Detergent screening of a G-protein-coupled receptor using serial and array biosensor technologies. Analytical Biochemistry 386: 98-104; (2009). Goto reference

This publication describes the benefits and limitations of two biosensor approaches for screening solubilization conditions for G-protein-coupled receptors (GPCRs). Assays designed for a serial processing instrument (Biacore 2000/3000/T100) and an array platform (Biacore Flexchip) were used to examine how effectively 96 different detergents solubilized the chemokine receptor CCR5 while maintaining its binding activity for a conformationally sensitive Fab (2D7). Using the serial processing instrument, the authors were able to analyze three samples in each 30-min binding cycle, thereby requiring approximately 24h to screen an entire 96-well plate of conditions. In contrast, with the array system, the characterization of all 96 detergents is done simultaneously, completing the assay in less than 1h. But the current array technology requires that the capture of the GPCR preparations off-line.