Development of a homogeneous heparanase assay using HTRF® technology

Materials: Human heparanase stable expression cell A375M lysate was kindly provided by Dr Higashi, of the University of Tokyo, Japan. Bovine kidney heparan sulfate (HS) was obtained from Seikagaku (Japan), 1-ethyl-3-(3-dimethylanmi nopropyl)carbodiimide HCl (EDC) and 5-(biotinamide)pentylamine were purchased from Pierce. Cryptate TBP mono- suberate and XL665-labeled streptavidin (SA-XL ent! ) were made at Cisbio international (France). Suramin, heparin, CHAPS, EIA grade BSA and potassium fl uoride (KF) were purchased from Sigma (USA). Preparation of biotin and cryptate labeled heparan sulfate substrate HS was fi rst biotinylated using EDC and 5-(biotinamide)pentylamine. Th e substrate was then labeled with europium cryptate TBP monosuberate. A mixture (0.3 ml) containing 20 mg/ml of HS, 3 mg/ml of 5-(biotinamide)pentylamine, 0.9 mg/ml of EDC and 0.1 M MES pH 6.0 was incubated at 25°C for 6 h. Aft er dialysis against 0.1 M NaPO4 pH 8.0 (10 kDa cut-off ), 3 mg of europium cryptate TBP monosuberate was added to the biotinylated HS solution and the mixture was incubated at 25°C for another 2 h. Aft er dialysis against PBS (10 kDa cut-off ), the HS concentration of biotin and cryptate labeled HS substrate (Biotin-HS-Eu(K)) was determined with the Blyscan glycosaminoglycan assay kit.