It is reported herein a new approach to study the orientation and density of mouse antibody 27 grafting on magnetic beads, serving for immunoassays and biosensors with fluorescent 28 detection of biomolecules. This approach is based on selective enzymatic digestion of target 29 grafted antibodies at a specific site below the hinge position to provide F(ab’)2 and Fc 30 fragments, followed by separation and determination of these fragments with size exclusion 31 chromatography (SEC) coupled with fluorescence detection (FLD). The developed method was 32 applied for evaluation of immunoglobulin (IgG2a) grafting capacity on three different 33 biofunctionalized magnetic beads (i.e., Tosyl-activated, carboxylic, protein G). Tosyl-activated 34 and protein G beads at different optimal grafting IgG: bead ratios (i.e.,110 μg: 1000 μg and 240 35 μg: 1000 μg, respectively) exhibited superior grafting capacity than carboxylic counterparts. 36 Under the optimized conditions, more than 70 % of antibodies were grafted on tosyl-activated 37 and protein G beads in the right orientation. This approach was then demonstrated with different 38 commercially available antibodies specific to amyloid-beta peptide 1-42 (Aβ1-42) for magneto-39 immunoassays and fluorescent detection of this peptide that is an established biomarker for 40 molecular diagnosis of Alzheimer’s disease.