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
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.
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.
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.
- 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
- Open Access
- Available under Open Access This content is freely available online to anyone, anywhere at any time.
- Online Date
- March 2013
- Online ISSN
- BioMed Central
- Additional Links
- Matrix metalloproteinase-9 activity
- Fluorescence resonance energy transfer peptide
- Immunocapture assay
- Focal cerebral ischemia
- Author Affiliations
- 1. Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL, 32610, USA
- 2. Department of Neurology, University of New Mexico Health Sciences Center, Albuquerque, NM, 87131, USA