Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is a homogeneous separation-based affinity method that enables the separation of preequilibrated ligands(L) and targets (T) from the L-T complexes based on the differences in their mobility. In the past, NECEEM has been used to study binding of protein to DNA complexes and more recently antibodies binding the antigen fluorescein, where the binding kinetics or mobilities facilitate separations. For broader diagnostic applications, it is beneficial to separate fluorescently labeled antibodies from their bound complexes. This is a more challenging analytical problem given the small differences in mobilities. In this study, we use fluorescently tagged anti β2-microglobulin (B2M), monoclonal antibody (Ab) and untagged B2M protein mixtures to perform an immunoassay to quantify the B2M protein in samples. B2M, is a protein present in the human serum which is used as a biomarker and a stage marker for Multiple Myeloma. The electrophoretic separation is carried out using short length, planar capillary electrophoresis platform that we have previously developed for rapid separations. Here, dual fluorescence and refractive index detection is implemented to monitor the separation. Equilibrated samples are injected onto the capillary and separated in under two minutes. The short length of the capillary (10 cm total length) enables fast separation times, which is essential to detect the bound complex before dissociation in the non-equilibrium conditions of the separation. Using this approach, not only can antigen levels be quantified, but also the dissociation constant and kinetic parameters. Progress towards developing this approach for immunoassays in patient samples will be presented.

Kinetic study of β2-microglobulin antigen-antibody complex using non-equilibrium capillary electrophoresis of equilibrium mixtures

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ACS Fall 2022

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