title: Fluorometric immunocapture assay for the specific measurement of matrix metalloproteinase-9 activity in biological samples: application to brain and plasma from rats with ischemic stroke.
abstract: BACKGROUND
  Matrix metalloproteinases are important factors in the molecular mechanisms leading to neuronal injury in many neurological disorders. Matrix metalloproteinase (MMP)-9 is up-regulated after cerebral ischemia and neuroinflammation and is actively involved in blood-brain barrier disruption. Current methods of measuring MMP-9 activity, such as gelatin-substrate zymography, are unspecific and arduous. Here we developed an immunocapture assay with high efficiency, specificity, and sensitivity for quantifying endogenously active as well as total MMP-9 activity.
  
  
  RESULTS
  A fluorescence resonance energy transfer (FRET) peptide-based immunocapture assay was developed that enables the accurate assessment of total and active forms of MMP-9 in complex biological samples. The FRET assay demonstrated correct and efficient binding of MMP-9 to a mouse monoclonal MMP-9 antibody and high specificity of the immunocapture antibody for MMP-9. Total and active levels of MMP-9 were measured in rat brain homogenates, plasma, human HT-1080 conditioned media, and RBE4 endothelial cell lysates. The FRET immunocapture assay yielded highly similar results for total MMP-9 activity when compared to gelatin-substrate zymography.
  
  
  CONCLUSIONS
  We suggest that the new FRET peptide-based immunocapture assay is a viable replacement of zymography for sensitive and high throughput quantification of MMP-9 activity in biological samples.
authors.affiliation: Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Kimberly E
authors.initials: KE
authors.lastname: Hawkins
authors.forename: Kelly M
authors.initials: KM
authors.lastname: DeMars
authors.forename: Changjun
authors.initials: C
authors.lastname: Yang
authors.forename: Gary A
authors.initials: GA
authors.lastname: Rosenberg
authors.forename: Eduardo
authors.initials: E
authors.lastname: Candelario-Jalil
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001921:Brain / Q000201:enzymology* / Q000473:pathology; D002545:Brain Ischemia / Q000097:blood* / Q000201:enzymology; D057075:Enzyme Assays / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D005470:Fluorometry / Q000379:methods*; D005780:Gelatin / Q000378:metabolism; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000097:blood* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D020521:Stroke / Q000097:blood* / Q000201:enzymology
chemical_list: D000911:Antibodies, Monoclonal; D010455:Peptides; D005780:Gelatin; D020780:Matrix Metalloproteinase 9