Active Melanoma Inhibitory Activity Protein 1 (MIA1)

Melanoma inhibitory activity (MIA), also known as cartilage-derived retinoic acid-sensitive protein (CD-RAP), is a 12-kDa protein that is secreted from both chondrocytes and malignant melanoma cells. MIA has been reported to have effects on cell growth and adhesion, and it may play a role in melanoma metastasis and cartilage development. In the presence of MIA, we observed enhanced migratory ability of melanocytic cells, induction of melanoma-associated genes as well as inhibition of apoptosis due to anoikis. S100 Calcium Binding Protein B (S100B) is a high affinity ligand for MIA1, a functional binding ELISA assay was conducted to detect the interaction of recombinant human MIA1 and recombinant bovine S100B. Briefly, MIA1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to S100B-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-MIA1 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, 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 450/630 nm immediately. The binding activity of recombinant humant MIA1 and recombinant bovine S100B was shown in Figure 1, the EC50 for this effect is 1.1 ug/mL.