On-Site Biolayer Interferometry-Based Biosensing of Carbamazepine in Whole Blood of Epileptic Patients

On-site monitoring of carbamazepine (CBZ) that allows rapid, sensitive, automatic, and high-throughput detection directly from whole blood is of urgent demand in current clinical practice for precision medicine. Herein, we developed two types (being indirect vs. direct) of fiber-optic biolayer interferometry (FO-BLI) biosensors for on-site CBZ monitoring. The indirect FO-BLI biosensor preincubated samples with monoclonal antibodies towards CBZ (MA-CBZ), and the mixture competes with immobilized CBZ to bind towards MA-CBZ. The direct FO-BLI biosensor used sample CBZ and CBZ-horseradish peroxidase (CBZ-HRP) conjugate to directly compete for binding with immobilized MA-CBZ, followed by a metal precipitate 3,3′-diaminobenzidine to amplify the signals. Indirect FO-BLI detected CBZ within its therapeutic range and was regenerated up to 12 times with negligible baseline drift, but reported results in 25 min. However, Direct FO-BLI achieved CBZ detection in approximately 7.5 min, down to as low as 10 ng/mL, with good accuracy, specificity and negligible matric interference using a high-salt buffer. Validation of Direct FO-BLI using six paired sera and whole blood from epileptic patients showed excellent agreement with ultra-performance liquid chromatography. Being automated and able to achieve high throughput, Direct FO-BLI proved itself to be more effective for integration into the clinic by delivering CBZ values from whole blood within minutes.

Characterization of new recombinant Immunoglobulins type-M binding to C1q by Biolayer Interferometry

The Immunoglobulins type-M (IgMs) are one of the first antibody classes mobilized during immune responses against pathogens and tumor cells. Binding to specific target antigens enables the interaction with the C1q complex which strongly activates the classical complement pathway. This biological function is the base for the huge therapeutic potential of IgMs but due to their high oligomeric complexity, in vitro production as well as biochemical and biophysical characterizations are challenging. In the present study, we present new attempts of recombinant production of two IgM models (IgM617 and IgM012) and the evaluation of their polymer distribution using biophysical methods (AUC, SEC-MALLS, Mass Photometry, transmission EM). Each IgM has an individual specific expression yield with different protein quality likely due to intrinsic IgM properties and patterning. Despite the presence of additional oligomeric states, purified recombinant IgMs retain their ability to activate complement in a C1q dependent manner. More importantly, a new method to evaluate their functional quality attribute by characterizing the kinetics of C1q binding to recombinant IgM has been developed using BioLayer Interferometry (BLI). We show that recombinant IgMs possess similar C1q binding properties as IgMs purified from human plasma.

Biolayer Interferometry for DNA-Protein Interactions v1

Biolayer interferometry (BLI) is a widely utilized technique for determining the interaction dynamics between macromolecules. Most BLI instruments, such as the Octet RED96e used throughout this protocol, are completely automated and detect changes in the interference pattern of white light reflected off a biosensor tip. Biosensors are initially loaded with a stationary macromolecule, then introduced into a solution containing macromolecules of interest. Binding to the stationary molecules creates a change in optical wavelength that is recorded by the instrument in real-time. The majority of published BLI experiments assess protein-protein (such as antibody-substrate kinetics) or protein-small molecule (such as drug discovery) interactions. However, a less-appreciated assay for BLI analysis is DNA-protein interactions. In our laboratory, we have shown the practicality of using biotinylated-DNA probes to determine the binding kinetics of transcription factors to specific DNA sequences. The following protocol describes these steps, including the generation of biotinylated-DNA probes, the execution of the BLI experiment, and data analysis through GraphPad Prism.