Focal Molography Allows for Affinity and Concentration Measurements of Proteins in Complex Matrices with High...

Characterizing biomolecular receptor-ligand interactions is critical for research and development. However, performing analyses in complex, biologically relevant matrices, such as serum, remains challenging due to non-specific binding that often impairs measurements. Here, we evaluated Focal Molography (FM) for determining K_D and kinetic constants in comparison to gold-standard methods using single-domain heavy-chain antibodies in various systems. FM provided kinetic constants highly comparable to SPR and BLI in standard buffers containing blocking proteins, with K_Ds of soluble CD4 (sCD4) interactions within a 2.4-fold range across technologies. In buffers lacking blocking proteins, FM demonstrated greater robustness against non-specific binding and rebinding effects. In serum, FM exhibited stable baseline signals, unlike SPR and BLI, and yielded K_Ds of sCD4 interaction in 50% Bovine Serum within a 1.8-fold range of those obtained in standard buffers. For challenging molecules prone to non-specific binding (Granzyme B), FM successfully determined kinetic constants without external referencing. Finally, FM enabled direct analyte quantification in complex matrices. sCD4 quantification in cell culture media and 50% FBS showed recovery rates of 97.8-100.3% with an inter-assay CV below 1.3%. This study demonstrates the high potential of FM for kinetic affinity determination and biomarker quantification in complex matrices, enabling reliable measurements under biologically relevant conditions.