High-throughput methods for measuring heparanase activity and screening potential antimetastatic and anti-inflammatory agents
| Autor: | Linda M. Reik, Debra Lucas-McGady, Chao-Min Liu, Kuo-Sen Huang, John Lawson Roberts, Janna Holmgren, Wayne Levin |
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| Rok vydání: | 2004 |
| Předmět: |
Fluorophore
Anti-Inflammatory Agents Drug Evaluation Preclinical Biophysics Biotin Gene Expression Antineoplastic Agents CHO Cells Kidney Tritium Fibroblast growth factor Biochemistry Extracellular matrix chemistry.chemical_compound Cricetinae Escherichia coli Animals Humans Heparanase Cloning Molecular Fluorescein isothiocyanate Molecular Biology Cells Cultured Glucuronidase Staining and Labeling Chemistry Substrate (chemistry) Cell Biology Molecular biology Fluorescence Fibroblast Growth Factors Cattle Heparitin Sulfate Fluorescein-5-isothiocyanate |
| Zdroj: | Analytical Biochemistry. 333:389-398 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2004.06.023 |
| Popis: | Heparanase plays an important role in the degradation of the extracellular matrix. It is implicated in inflammation, tumor angiogenesis and metastasis. We have developed two high-throughput methods for measuring heparanase activity and screening potential inhibitors. The first method involves coating fibroblast growth factor (FGF) on microtiter plates and capturing fluorescein isothiocyanate (FITC)-labeled heparin sulfate (HS), which is used as a substrate for heparanase digestion. Labeled HS fragments are released into the medium and quantitated by fluorescence intensity measurement. We have implemented this assay method into a Zeiss uHTS system and screened compound libraries for heparanase inhibitors. The second method involves labeling HS with biotin followed by FITC to generate a dual-labeled HS. The labeled material is bound to streptavidin-coated plates and used as a substrate for heparanase digestion. Both methods are sensitive and easily applicable to robotic systems. In addition, we have labeled both HS and biotin-HS with Eu-chelate, a fluorophore that exhibits long decay fluorescence. Assays using Eu-labeled HS and Eu-labeled biotin-HS have been developed and show higher sensitivity than those using FITC-labeled material. Furthermore, assays using Eu-chelate HS (or biotin-HS) should eliminate the interference of fluorescence compounds. |
| Databáze: | OpenAIRE |
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