Antibody Profiling: Kinetics with Native Biomarkers for Diagnostic Assay and Drug Developments

Abstract

Despite remarkable progress in applied Surface Plasmon Resonance (SPR)-based methods, concise monitoring of kinetic properties for native biomarkers from patient samples is still lacking. Not only are low concentrations of native targets in patient samples, often in the pM range, a limiting and challenging factor, but body fluids as complex matrices furthermore complicate measurements. The here-described method enables the determination of kinetic constants and resulting affinities for native antigens from patients’ cerebrospinal fluid (CSF) and sera binding to antibodies. Using a significantly extended target-enrichment step, we modified a common sandwich-assay protocol, based on a primary and secondary antibody. We successfully analyze antibody kinetics of native targets from a variety of origins, with consistent results, independent of their source. Moreover, native neurofilament light chain (NFL) was investigated as an exemplary biomarker. Obtained data reveal antibodies recognizing recombinant NFL with high affinities, while showing no, or only significantly weakened binding to native NFL. The indicated differences for recombinant vs. native material demonstrate another beneficial application. Our assay is highly suitable for gaining valuable insights into characteristics of native biomarkers, thus impacting on the binder development of diagnostic reagents or pharmaceutical drugs.