| Assay Method Information | |
| | Heparanase Inhibition Assay |
| Description: | Recombinant human active heparanase derived from Chinese hamster ovary cells was from R&D Systems. Bovine serum albumin-coated 96 well microplates (96F Maxisorp NNC #456537, Thermo Scientific) were used for the assays and were prepared by incubation of the plates with 1% (w/v) BSA dissolved in phosphate-buffered saline containing 0.05% (v/v) Tween-20 (PBST) at 37° C. for 1 h. The plates were washed three times with PBST, shaken dry, and stored for up to two weeks at 4° C. before use. Assay mixtures typically contained 42.5 mM sodium acetate buffer (pH 5.0), 0.8 nM heparanase, 100 μM fondaparinux (Arixtra , Aspen Pharmacare), 5% (v/v) DMSO, and varying concentrations of inhibitor in a total volume of 100 μL. Following initiation of the reaction by addition of fondaparinux, the plate was sealed with adhesive film and incubated at 37° C. for 20-24 h. 100 μL of 1.69 mM WST-1 (Dojindo) solution in 0.1 M NaOH was added to the assay mixture. The plate was resealed and developed by incubation at 60° C. for 1 h, and the absorbance was measured at 584 nm. For the enzymatic assays, test compounds were dissolved and added to the assay at varying concentrations to calculate the level of inhibition. |
| Affinity data for this assay | |
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