Transferrin, an iron binding protein that facilitates iron uptake in cells, is an integral part of a mechanism that may introduce antibody therapies to the central nervous system. Currently, the brain’s ability to take in antibody therapies is limited by the presence of the blood brain barrier. While novel mechanisms to manipulate the transfer of important proteins and antibodies across the BBB have been tested, endogenous receptor-mediated transcytosis (RMT) is one mechanism of particular interest. Iron transfer is achieved when transferrin bound iron connects with the transferrin receptor, and iron subsequently enters the cell by way of clathrin-mediated endocytosis. Once inside the cell, transferrin is trafficked to early endosomes, where the iron is dropped off. After iron delivery, transferrin is directed to recycling endosomes to be brought back to the cell surface. Using this pathway to deliver therapeutic antibodies across the BBB has shown both failures and successes, and the application of the Transferrin Receptor antibody (OX26) in the following articles take a closer look at this process.
TfR (Transferrin R) Antibody (OX26) [NB200-585] - PC-12 cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton-X100. The cells were incubated with anti TfR/Transferrin R (OX26) NB200-585 at a 1:100 dilution overnight at 4C and detected with an anti-mouse Dylight 488 (Green) at a 1:500 dilution. Actin was detected with Phalloidin 568 (Red) at a 1:200 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.
The Transferrin Receptor antibody (OX26) was used in Moos et al in order to study transferrin uptake across the BBB in rats. This research was primarily carried out using brain capillary endothelial cells (BCECs), which compose most of the BBB. To circumvent the BBB, antibodies were raised against peptides expressed by BCEC’s, and the Transferrin Receptor antibody (OX26) is specific to the BCEC transferrin receptor. Transferrin and iron transport were measured in both developing and adult rats, using a non-immune IgG2a as a marker of non-specific transport. Following successful uptake, the Transferrin Receptor antibody (OX26) was detected in brain capillaries of the brain parenchyma and a Transferrin Receptor antibody (OX26) was used in immunohistochemistry for further analysis. Overall, results determined that facilitated uptake of OX26 is successful in the brain and liver. Furthermore, there was a correlation between age and transferrin uptake.
Another study from Gosk et al investigated the targeted transfer of the Transferrin Receptor antibody (OX26) when conjugated to polyethyleneglycol-coated liposomes in 18-day-old rats. First, the Transferrin Receptor antibody (OX26) was used in immunohistochemistry to visualize the distribution of transferrin receptors in BCECs. The OX26 was found to localize throughout the CNS, aside from the surrounding organs. Over 80% of the radiolabeled OX26 was found at the BCECs, whereas neither OX26 nor immunoliposomes stained nearby neuronal or glial cell populations. Overall, it is clear that receptor mediated transferrin uptake is a successful method of transferring liposomes and other cellular cargo into BCECs. However, determining a method to transfer cargo deeper into the BBB has yet to be discovered with the transferrin receptor pathway.
Novus Biologicals offers TfR (Transferrin R) reagents for your research needs including:
