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.
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.
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DOI：
10.1186/1756-6606-6-14
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发表时间：
2013-03-23
期刊：
影响因子：
3.6
通讯作者：
Candelario-Jalil E
中科院分区：
文献类型：
--
作者：
Hawkins KE;DeMars KM;Yang C;Rosenberg GA;Candelario-Jalil E
基质金属蛋白酶是许多神经系统疾病中导致神经元损伤的重要分子机制。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，是非特异性和艰巨的。在这里，我们开发了一种高效，特异性和灵敏度的定量内源性活性以及总MMP-9活性的免疫捕获测定。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，该测定法能够准确评估复杂生物样品中MMP-9的总形式和活性形式。FRET测定证明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的高特异性。在大鼠脑匀浆、血浆、人HT-1080条件培养基和RBE 4内皮细胞裂解物中测量MMP-9的总水平和活性水平。FRET免疫捕获试验产生了高度相似的结果，总MMP-9活性相比，明胶底物酶谱。我们认为，新的FRET肽为基础的免疫捕获试验是一个可行的替代酶谱法的灵敏和高通量定量生物样品中的MMP-9活性。
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.
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影响因子：
8.3
作者：
Candelario-Jalil E;Thompson J;Taheri S;Grossetete M;Adair JC;Edmonds E;Prestopnik J;Wills J;Rosenberg GA
通讯作者：
Rosenberg GA
DOI：
10.1097/00004647-199906000-00005
发表时间：
1999-06-01
影响因子：
6.3
作者：
Heo, JH;Lucero, J;del Zoppo, GJ
通讯作者：
del Zoppo, GJ
DOI：
10.1097/01.wcb.0000100044.07481.97
发表时间：
2003-12-01
影响因子：
6.3
作者：
Montaner, J;Rovira, A;Alvarez-Sabín, J
通讯作者：
Alvarez-Sabín, J
影响因子：
5.2
作者：
Lee, M;Bernardo, MM;Mobashery, S
通讯作者：
Mobashery, S
影响因子：
4.8
作者：
Fu, XY;Kassim, SY;Heinecke, JW
通讯作者：
Heinecke, JW