Drug delivery to the central nervous system remains an impeding obstacle for the development of biopharmaceuticals. To study this, targeting strategies by genetic fusion to a molecular Trojan horse are used by utilizing receptor-mediated transcytosis, such as those governed by the transferrin receptor. Even though this approach has been effective, limiting factors related to how antibodies bind with transferrin receptor has not been readily defined. To study this, a single chain fragment constant (Fc) conjugated to a scFv8D3 (scFc-scFv8D3) and a chimeric mouse IgG 8D3 were designed and produced by Morrison et al (2023). Using living microvascular endothelial cells expressing transferrin receptors, antibody binding was analyzed using LigandTracer. The kinetic rate constants ka (association rate constant), kd (dissociation rate constant) were extracted for scFc-scFv8D3 and the chimeric mouse IgG 8D3 using a 1:1 model with distinct differences in binding stability (kd). The chimeric mouse IgG 8D3 exhibited a KD (equilibrium dissociation constant) of 0.1 nM, with ka (association rate constant) of 3.38×104 (M-1 s-1) and kd (dissociation rate constant) of 3.72×10-6 (s-1). The fitting of scFc-scFv8D3 yielded a KD of 2.96 nM, ka 1.69×104 (M-1 s-1), and a faster kd of 5.49×10-5 (s-1). To gain insight into the intracellular behaviour of scFc-scFv8D3 and the chimeric mouse IgG 8D3, a pH-sensitive dye, pHrodo GreenTM succinimidyl ester (ThermoFisher) was conjugated to the antibodies. With the LigandTracer in 37°C, it was observed that the chimeric mouse IgG 8D3 (bivalent) significantly accumulated in an acidic milieu, characteristic of the late endosome or lysosome. This result is consistent with the idea of Transferrin-receptor cross-linking inducing a degradation pathway. The scFc-scFv8D3 (monovalent) internalization suggests a re-direction of transcellular trafficking away from an acidic milieu. These findings imply that the affinity and mode of binding of antibodies play a crucial role in determining their intracellular fate and trafficking pathways.