Immuno-capture of biomolecules on functionalized magnetic beads: From characterization to fluorescent detection of a biomarker for Alzheimer's disease toward high performance biosensing

It is reported herein a new approach to study the orientation and density of mouse antibody grafting on magnetic beads, serving for immunoassays and biosensors with fluorescent detection of biomolecules. This approach is based on selective enzymatic digestion of target grafted antibodies at a specific site below the hinge position to provide F(ab')2 and Fc fragments, followed by separation and determination of these fragments with size exclusion chromatography (SEC) coupled with fluorescence detection (FLD). The developed method was applied for evaluation of immunoglobulin (IgG2a) grafting capacity on three different biofunctionalized magnetic beads (i.e., Tosyl-activated, carboxylic, protein G). Tosyl-activated and protein G beads at different optimal grafting IgG: bead ratios (i.e.,110 µg: 1000 µg and 240 µg: 1000 µg, respectively) exhibited superior grafting capacity than carboxylic counterparts. Under the optimized conditions, more than 70 % of antibodies were grafted on tosyl-activated and protein G beads in the right orientation. This approach was then demonstrated with different commercially available antibodies specific to amyloid-beta peptide 1-42 (Aβ1-42) for magneto-immunoassays and fluorescent detection of this peptide that is an established biomarker for molecular diagnosis of Alzheimer's disease.