Methodology

Molecular Brain

, 6:14

Open Access This content is freely available online to anyone, anywhere at any time.

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

  • Kimberly E HawkinsAffiliated withDepartment of Neuroscience, McKnight Brain Institute, University of Florida
  • , Kelly M DeMarsAffiliated withDepartment of Neuroscience, McKnight Brain Institute, University of Florida
  • , Changjun YangAffiliated withDepartment of Neuroscience, McKnight Brain Institute, University of Florida
  • , Gary A RosenbergAffiliated withDepartment of Neurology, University of New Mexico Health Sciences Center
  • , Eduardo Candelario-JalilAffiliated withDepartment of Neuroscience, McKnight Brain Institute, University of Florida Email author 

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.

Keywords

Matrix metalloproteinase-9 activity Fluorescence resonance energy transfer peptide Immunocapture assay Focal cerebral ischemia