Active Collagen Type IV Alpha 1 (COL4a1)

Collagen Type IV Alpha 1 (COL4a1), a secreted glycoprotein ,is a  member of the type IV collagen family. COL4a1 is the major structural component of glomerular basement membranes (GBM), forming a 'chicken-wire' meshwork together with laminins, proteoglycans and entactin/nidogen. It can inhibit angiogenesis and tumor formation, and specifically inhibit endothelial cell proliferation, migration and tubule formation. COL4A1 mutations can remain asymptomatic or cause devastating disease. Neonates and children may present with porencephaly, intracerebral hemorrhage, or hemiparesis, whereas adults tend to develop intracranial aneurysms or retinal arteriolar tortuosities. Glycoprotein VI (GP6) has been identified as an interactor of COL4a1, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human COL4a1 and recombinant human GP6. Briefly, COL4a1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to GP6-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-COL4a1 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 human COL4a1 and recombinant human GP6 was shown in Figure 1, the EC50 for this effect is 0.05 ug/mL.