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14 BioLayer Interferometry BioLayer Interferometry (BLI) is a label-free, biosensor-based technology for measuring biomolecular interactions. Even though BLI is similar to SPR in many ways, there are also crucial differences. Conclusion Although BLI is less sensitive than SPR, it's easier to use. It's a great tool for mAb development workflows. There are options for low, medium and higher throughput sample analysis. How it works BLI is an optical technique that relies on the physical phenomenon of interference. One binding partner, the ligand, is immobilized on a biosensor. The biosensor tip is dipped into a solution containing the other binding partner (the analyte), which starts binding, creating another layer on the biosensor. White light is then directed at the biosensor tip and reflects off this new layer as well as the biosensor surface. The way the light reflects off of both layers causes interference, and the interference pattern changes with the amount of analyte bound to the biosensor tip. This signal is read in real time and provides detailed information regarding the kinetics of association and dissociation of the two molecules as well as the affinity constant for the interaction (k on , k off and K d ). Strengths Its ease of use and throughput make it an attractive option, especially for labs developing monoclonal antibodies (mAbs). Due to the biosensor tip design and the absence of microfluidic tubing in contrast to SPR, the technique is highly amenable to both purified and crude samples, and there is no need for flushing and cleaning steps. Analysis of kinetics (k on , k off ) and affinity can be performed. The method can also be used to determine the molar concentration of analytes. Measurements are fast as it is a "dip and read" methodology. Weaknesses BLI is less sensitive and less robust than SPR, which makes it poorly suited for applications examining small-molecule interaction partners. This, however, does not apply for most mAb applications. As with SPR, covalent coupling of biomolecules to the biosensor is difficult and faces the same hurdles in terms of immobilization conditions. Moreover, it is hardly possible to achieve identical ligand coatings of multiple biosensor tips, so that regeneration of a tip is still required if concentration series are measured. Especially for high-affinity interactions, the mass transfer limitation is an important factor, as well as the limitation in measurement times due to sample evaporation from the plate.

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