Active Vascular Cell Adhesion Molecule 1 (VCAM1)

Vascular cell adhesion protein 1 (VCAM-1) is a cell adhesion molecule and a member of the immunoglobulin superfamily. Alternatively spliced forms are known to occur, but the most common form is a type I transmembrane protein with a 674 amino acid extracellular segment that includes seven C2-type immunoglobulin domains. VCAM-1 is expressed by activated endothelial cells and certain other cell types including macrophages, dendritic cells, neurons, smooth muscle cells, fibroblasts, and oocytes. Besides, Integrin Alpha 4 (ITGa4) has been identified as an interactor of VCAM-1, thus a binding ELISA assay was conducted to detect the interaction of recombinant human VCAM-1 and recombinant human ITGa4. Briefly, biotin-linked VCAM-1 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to ITGa4-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 450nm immediately. The binding activity of recombinant human VCAM-1 and recombinant human ITGa4 was shown in Figure 1, the EC50 for this effect is 0.261 ug/mL.

As VCAM1 has the function of cell adhesion, we measure the activity of VCAM1 by the ability of the immobilized protein to support the adhesion of U937 human histiocytic lymphoma cells. When 5 x 104 cells/well are added to recombinat human VCAM1 coated plates (2.5 µg/mL with 100 µL/well), approximately 80-90% will adhere after 3 hour incubation at 37℃. The adhesion of U937 after 3 hour incubation at 37℃ observed by inverted microscope was shown in Figure 2.