Comparison of the Association (A), Dissociation (B), and Equilibrium (C) Binding Constants Generated by the Four Biosensor Platforms. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical theory, with fiber-optic probes Proscillaridin A acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on Proscillaridin A continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation. Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments’ ability to provide quality Proscillaridin A kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular conversation model. Keywords: Biochemistry, Issue 122, optical biosensors, antibody-antigen interactions, binding kinetics, drug discovery, Surface Plasmon Resonance, BioLayer Interferometry at different mAb surface densities, and Physique 9 further compares the calculated binding activities of the mAb surfaces across the biosensor platforms. Physique 1. Multiplex Ligand Arrays of Amine-coupled (A) and Fc-captured (B) Antibody Surfaces from CFM Printing in the IBIS MX96. Images of the printed arrays are shown in the panels, where the grey areas enclosed by red squares indicate the presence of antibody. The darker interspots located between the active antibody spots are used for reference subtraction. Each column contains an antibody printed at the titration concentrations identified below and to the left of the image. The amounts of the printed antibodies quantified by calculating the difference in bulk shifts between the active and reference locations are shown in the panels), medium- (panels), and low- (panels) density surfaces. The overlaid easy black lines represent the kinetic fit of the binding response signals at different human PCSK9 concentrations (colored lines) to a 1:1 conversation model. Please click here to view a larger version of this figure. Physique 4. Binding Sensorgrams of the Captured Antibodies Interacting with Human PCSK9 and 1:1 Kinetic Model Fit Overlays in the ProteOn XPR36. The interactions are evaluated over high- (panels), medium- (panels), and low- (panels) density surfaces. The overlaid easy black lines represent the kinetic fit of the binding response signals at different human PCSK9 concentrations (colored lines) to a 1:1 conversation model. Please click here to view a larger version of this figure. Physique 5. Binding Sensorgrams of the Captured Antibodies Interacting with Human PCSK9 and 1:1 Kinetic Model Fit Overlays in the Octet RED384. The interactions are evaluated over high- (panels), medium- (panels), and low- (panels) density surfaces. The overlaid red lines represent the kinetic Proscillaridin A fit of the binding response signals at different human PCSK9 concentrations (colored lines) to a 1:1 conversation model. Please click here to view a larger version of this figure. Physique 6. Binding Sensorgrams of the Amine-coupled (A) and Fc-captured (B) Antibodies Interacting with Human PCSK9 and 1:1 Kinetic Model Fit Overlays in the IBIS MX96. The binding profiles are organized as 10 x 8 panels that follow the array plate map in Rabbit Polyclonal to p15 INK Physique 1. The black lines represent the recorded binding response signals at different human PCSK9 concentrations, and the overlaid red lines represent the fitted curves. Please click here to view a larger version of this figure. Physique 7. Comparison of the Association (A), Dissociation (B), and Equilibrium (C) Binding Constants Generated by the Four Biosensor Platforms. The kinetic parameters are derived from global analysis of the binding curves in Figures 3 – 6. The devices are represented as follows: Biacore T100 (blue), ProteOn XPR36 (green), Octet RED384 (red), IBIS MX96, amine-coupled (purple), and IBIS MX96, Fc-captured (orange). Please click here to view a larger version of this physique. Figure 8. Comparison of the Consistency of Kinetic Rate Constants Over Multiple Antibody Surface Densities in the Biacore T100 (A), ProteOn XPR36 (B), Octet RED384 (C), IBIS MX96, amine-coupled (D), and IBIS MX96, Fc-captured (E). The kinetic parameters (sub-panels), (sub-panels), and (in the hundreds or thousands), for off-rate ranking/kinetic.