Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA.
Mol Brain. 2013 Mar 23;6:14. doi: 10.1186/1756-6606-6-14.
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.
基质金属蛋白酶是导致许多神经紊乱中神经元损伤的分子机制中的重要因素。基质金属蛋白酶（MMP）-9 在脑缺血和神经炎症后上调，并积极参与血脑屏障破坏。目前测量 MMP-9 活性的方法，如明胶底物酶谱法，不具有特异性且繁琐。在这里，我们开发了一种高效、特异和灵敏的免疫捕获测定法，用于定量内源性活性和总 MMP-9 活性。
开发了一种基于荧光共振能量转移（FRET）肽的免疫捕获测定法，可准确评估复杂生物样品中的总和活性形式的 MMP-9。FRET 测定法证明了 MMP-9 与小鼠单克隆 MMP-9 抗体的正确和有效结合，以及免疫捕获抗体对 MMP-9 的高度特异性。在大鼠脑匀浆、血浆、人 HT-1080 条件培养基和 RBE4 内皮细胞裂解物中测量了总 MMP-9 和活性 MMP-9 的水平。与明胶底物酶谱法相比，FRET 免疫捕获测定法在总 MMP-9 活性的测定中产生了高度相似的结果。
我们认为，新的基于 FRET 肽的免疫捕获测定法是一种可行的替代酶谱法，可用于生物样品中 MMP-9 活性的敏感和高通量定量。
