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[JOVE] Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platfor


Abstract:

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. 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 principle, with fiber-optic probes 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 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 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 interaction model.

Danlin Yang1, Ajit Singh2, Helen Wu1, Rachel Kroe-Barrett1

1Department of Biotherapeutics Discovery, Immune Modulation and Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals, Inc., 2The Fu Foundation School of Engineering and Applied Science, Columbia University

Link: https://www.jove.com/video/55659/determination-high-affinity-antibody-antigen-binding-kinetics-using

#04292017 #cell #labelfree #drugdiscovery #antibody #biosensor #MEMS #labonachip #microchannels

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