CD320, also known as the Transcobalamin II receptor (TCb1R) and 8D6A, is an approximately 60 kDa transmembrane member of the LDL receptor family. CD320 is expressed in virtually all tissues and is tightly regulated according to the proliferative/differentiation status of the cell and the cellular levels of B12. Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320. Cubilin (CUBN) is the intrinsic factor-vitamin B12 receptor, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human CD320 and recombinant human CUBN. Briefly, biotin-linked CD320 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to CUBN-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 µl stop solution to the wells and read at 450 nm immediately. The binding activity of recombinant human CD320 and recombinant human CUBN was shown in Figure 1, the EC50 for this effect is 0.4 ug/mL.