A novel and simple immunocapture assay for determination of gelatinase-B (MMP-9) activities in biological fluids: Saliva from patients with Sjogren's syndrome contain increased latent and active gelatinase-B levels
A novel and simple immunocapture assay for determination of gelatinase-B (MMP-9) activities in biological fluids: Saliva from patients with Sjogren's syndrome contain increased latent and active gelatinase-B levels
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DOI：
10.1016/s0945-053x(98)90116-0
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发表时间：
1998-12-01
期刊：
影响因子：
6.9
通讯作者：
Verheijen, JH
中科院分区：
文献类型：
--
作者：
Hanemaaijer, R;Visser, H;Verheijen, JH
在这里，我们描述了一种新的原理，通过比色法获得人基质金属蛋白酶（MMPs）不同成员的活性。利用蛋白质工程技术，制备了一种修饰的前尿激酶，其中通常被纤溶酶识别的激活序列（ProArgPheLys向下箭头IleIleGlyGly）被MMPs特异性识别的序列（ArgProLeuCly向下箭头IleIleClyGly）所取代。由这种修饰的前尿激酶被MMPs激活而产生的活性尿激酶可以用尿激酶显色肽底物直接测量。该试验使用MMP-9特异性单克隆抗体对MMP-9进行特异性检测。使用该抗体从生物液体或组织培养基中捕获MMP-9，并分析活性和潜伏MMP-9的mmp -活性。我们测定了干燥综合征患者唾液中明胶酶b （MMP-9）的活性。使用放射性标记的凝胶化胶原进行一般明胶酶测定，观察到明胶酶活性在患者中略有增加，但不显著：与健康对照组相比，患者的一般明胶酶活性分别为：17.0 +/- 4.9 vs 12.2 +/- 2.5 × 10(4) cpm/ml (p > 0.05)，活性和潜在明胶酶分别为44.0 (4.0 vs 36.1 +/- 1.9 × 10(4) cpm/ml （p > 0.05）。然而，使用免疫捕获活性测定（使用改良尿激酶）特异性测量了MMP-9活性，与健康对照组相比，患者唾液中的MMP-9活性显着增加：MMP-9（已经活性）：患者8.9 +/- 2.5 U/mg，对照组1.0 +/- 0.5 U/mg (p = 0.002)；潜伏+活性MMP-9：患者53.1 +/- 9.8 U/mg，对照组16.5 +/- 2.6 U/mg （p = 0.01）。该检测使用改良的前尿激酶作为底物来测量MMP-9活性，可以很容易地适用于mmp -家族的各种成员或其他难以测量的蛋白酶的特定检测，其格式可用于高通量筛选化合物或样品。
Here we describe a new principle for accessing the activity of the different members of the human matrix-metalloproteinases (MMPs) by a colorimetric assay. Using protein engineering, a modified pro-urokinase was made in which the activation sequence, normally recognized by plasmin (ProArgPheLys down arrow IleIleGlyGly), was replaced by a sequence that is specifically recognized by MMPs (ArgProLeuCly down arrow IleIleClyGly). The active urokinase resulting from the activation of this modified pro-urokinase by MMPs can be measured directly using a chromogenic peptide substrate for urokinase. The assay has been made specific for MMP-9 using an MMP-9 specific monoclonal antibody. Using this antibody MMP-9 is captured from biological fluids or tissue culture media, and MMP-activity of both active and latent MMP-9 can be analysed.We determined the gelatinase-B (MMP-9) activity present in saliva from patients with Sjogren's syndrome. Using a general gelatinase assay with radioactively-labeled gelatinated collagen it was observed that gelatinase activity was slightly, though not significantly, increased in patients: general gelatinase activity in patients versus healthy controls: 17.0 +/- 4.9 vs 12.2 +/- 2.5 x 10(4) cpm/ml (p > 0.05, and 44.0 ( 4.0 vs 36.1 +/- 1.9 x 10(4) cpm/ml (p > 0.05), for active and latent gelatinase, respectively. However using the immunocapture activity assay (using modified urokinase) specifically MMP-9 activity was measured, which was significantly increased in saliva from patients compared to healthy controls: MMP-9 (already active): patients 8.9 +/- 2.5 U/mg, controls 1.0 +/- 0.5 U/mg (p = 0.002); latent plus active MMP-9: patients 53.1 +/- 9.8 U/mg, controls 16.5 +/- 2.6 U/mg (p = 0.01).This assay, measuring MMP-9 activity using modified pro-urokinase as a substrate can easily be adapted for the specific detection of the various members of the MMP-family or other difficult to measure proteases, in a format that can be used for high throughput screening of compounds or samples.