While LT-71 and MEM-75 showed different degrees of transcytosis and recycling. Although these data also show a correlation between affinity and transcytosis, the comparable affinities of antibodies M-A712 and MEM-189, combined to their strikingly different transcytosis behavior, indicate that there may be additional mechanisms governing the intracellular fate of transcytosing antibodies. Finally, we wanted to investigate if transcytosis of pH-dependent TfR antibodies could be blocked by bafilomycin, an inhibitor of endosomal acidification. Figure 6I shows that pre-incubation of hCMEC/D3 cells with bafilomycin strongly reduced basolateral passage of antibody MEM-189, while apical recycling was unaffected. This result confirmed our hypothesis that endosomal acidification is an essential mechanistic step in facilitating the transcytosis of TfR antibodies with reduced affinity at low pH. There is a wealth of literature describing the phenomenon of transcytosis in vivo and in vitro in addition to the classical pathway of receptor-mediated endocytosis and recycling. However, many publications describing in vitro models of protein transcytosis through the blood-brain barrier neglect the magnitude of paracellular flux as opposed to the small amount of transcytosed material. In fact, our results show accumulation of 30 ng of the transferrin ligand or Protein A in the basolateral compartment following incubation with 1 mg/mL of the radiolabel. In order to cope with this limitation, which is especially apparent in the relatively leaky monolayers of hCMEC/D3 cells, we have applied a pulse-chase assay set-up, initially described by Raub and Newton for primary bovine brain endothelial cells. In order to detect the low amounts of transcytosed antibody and at the same time avoid using radiolabeled material, the development of highly sensitive IgG ELISAs has been instrumental for assessing the transcytosis potential of antibodies of different species. Furthermore, the ELISA protocol can be easily adapted for automation which makes it highly attractive in terms of assay throughput. Although several studies addressed the transcytosis of antibodytargeted nanoparticles and immunoliposomes across hCMEC/D3, this is the first study to investigate the transcytosis and the fate of free antibodies targeting receptors capable of mediating transcytosis in immortalized human brain endothelium. Our results support the following conclusions: Apical to basolateral transcytosis of intact transferrin occurs in hCMEC/D3 monolayer cultures. Ligand is also equally recycled to the luminal membrane. Both events are temperature sensitive but not modulated by astrocyte co culture An antibody to the IGF1R is exclusively recycled, while antibodies against the TfR are either degraded in lysosmes or recycled/transcytosed Reduced affinity of antibodies to the transferrin receptor at endosomal pH may enhance antibody transcytosis.