Malachite green assay

Example 10

B cells were isolated from human leukopak using EasySep B cell isolation kit (STEMCELL Technologies). 5×10⁴ B cells/well were washed with Tris buffer and incubated with serially diluted (100-0.00013 nM) antibodies for 30 minutes at 37 degrees C. 50 μM ATP was added to each well and incubated with cells for 2 hrs. The supernatants were collected and analyzed in Malachite Green Assay (R&D) according to manufacturer's protocol. Phosphate released from CD39 processing of ATP was used as a readout of enzyme activity. Palivizumab was used as an isotype control and ARL (Tocris) and POM-1 (Alpha Aesar), non-specific small molecule inhibitors of CD39, were used as positive controls at 100 μM.

The antibodies were demonstrated to bind to primary human and cyno B cells (see FIG. 7) and the next step was to evaluate the inhibition of ATP hydrolysis by detection of free phosphate (Pi) using a Malachite Green Assay. The results are shown in FIG. 8 and indicate the anti-CD39 antibodies inhibit the enzymatic inhibition/dephosphorylation of ATP by primary human B cells. The ability of the antibodies to inhibit enzymatic activity was comparable regardless of high vs. low max MFI detected in the binding to human B cells (FIG. 7).

Example 11

Human monocytes were purified from leukopak using EasySep Human monocytes isolation kit (STEMCELL). Cyno monocytes were isolated from whole cyno blood using NHP CD14 positive selection kit (Miltenyi). Monocytes at 5×10⁴ cells/well were washed with Tris buffer and incubated with serially diluted (100-0.00013 nM) anti-CD39 antibodies for 30 minutes at 37 C. 50 μM ATP was added to the cells for 15 minutes at 37 C and supernatants were harvested and analyzed in Malachite Green Assay (R&D) for phosphate levels. Palivizμmab was used as an isotype control and ARL (Tocris) and POM-1 (Alpha Aesar), non-specific small molecule inhibitors of CD39, were used as positive controls at 100 μM.

CD39 expression has been detected on human leukocytes with the highest expression detected on monocytes (Thromb Res. 2007; 121(3):309-17). Because of this information, it was important to evaluate the ability of the anti-CD39 antibodies to inhibit ATPase activity on the cell surface. As demonstrated in FIG. 7, anti-CD39 antibodies bind to both human and cyno B cells. It is appropriate to evaluate the inhibition of enzymatic activity on human and cyno monocytes. The results indicate that all the antibodies are able to inhibit ATPase activity of CD39 on human and cyno monocytes with similar potencies.