Doughty J R, Goldberg R L, Ganu V, Melton R A, Hu S I, Di Pasquale G
Research Department, CIBA-GEIGY Corp., Summit, NJ 07901.
Agents Actions. 1993;39 Spec No:C151-3. doi: 10.1007/BF01972750.
Human proteoglycan was aggregated to an immobilized hyaluronan solid phase on a 96-well ELISA plate. This complex was then degraded by recombinant human stromelysin. The remaining proteoglycan fragments were detected using a monoclonal antibody probe directed against the chondroitin sulfate (CS) region of the core protein. Stromelysin degraded the aggregate in a time and dose dependent manner as reflected by the loss of the CS epitope. Assay sensitivity was 0.125 U/well with total loss of the CS epitope occurring at 4 U/well. o-phenanthroline (IC50 = 52 microM) and U24522 (IC50 = 9 microM) inhibited degradation, while phosphoramidon did not. Serine and cysteine protease inhibitors had no effect. A comparative analysis of this assay with a reference method, substance P assay, gave similar inhibitor profiles. The use of aggregated human proteoglycan (native conformation) as a substrate, may better reflect how stromelysin inhibitors behave in the presence of complex substrates such as cartilage matrix.
人蛋白聚糖与96孔酶联免疫吸附测定（ELISA）板上固定化的透明质酸固相聚集。然后，该复合物被重组人基质溶素降解。使用针对核心蛋白硫酸软骨素（CS）区域的单克隆抗体探针检测剩余的蛋白聚糖片段。如CS表位的丧失所反映，基质溶素以时间和剂量依赖的方式降解聚集物。测定灵敏度为0.125 U/孔，在4 U/孔时CS表位完全丧失。邻菲啰啉（IC50 = 52 μM）和U24522（IC50 = 9 μM）抑制降解，而磷酰胺素则无此作用。丝氨酸和半胱氨酸蛋白酶抑制剂无作用。该测定法与参考方法（P物质测定法）的比较分析给出了相似的抑制剂谱。使用聚集的人蛋白聚糖（天然构象）作为底物，可能更好地反映基质溶素抑制剂在诸如软骨基质等复杂底物存在下的行为。
