Fig. 6. Antibody 733 inhibits heparanase enzymatic activity. (A) Purified, active recombinant heparanase (20 ng) was added to 1 ml RPMI medium and incubated with affinity-purified antibody 733 (10 µg, red symbols), or rabbit IgG (black symbols) for 1 hour on ice, followed by 1 hour incubation with ³⁵S-labeled ECM. Heparanase activity was determined as described in Materials and Methods. (B) Heparanase-transfected 293 cells (2x10⁵) were plated on ³⁵S-labeled ECM in the presence of affinity-purified antibody 733 (30 µg/ml, red symbols) or control rabbit IgG (black symbols) for 2 hours. The incubation medium containing sulfate-labeled degradation fragments was subjected to gel filtration on a Sepharose CL-6B column.