Immunocapture-based fluorometric assay for the measurement of neprilysin-specific enzyme activity in brain tissue homogenates and cerebrospinal fluid
Immunocapture-based fluorometric assay for the measurement of neprilysin-specific enzyme activity in brain tissue homogenates and cerebrospinal fluid
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DOI：
10.1016/j.jneumeth.2007.08.012
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发表时间：
2008-01-30
影响因子：
3
通讯作者：
Love, Seth
中科院分区：
文献类型：
--
作者：
Miners, James Scott;Verbeek, Marcel M.;Love, Seth
脑啡肽酶是一种锌金属内肽酶，在高血压、癌症和阿尔茨海默病等多种疾病的生理和病理中具有重要作用。我们已经开发了一种免疫捕获法来测量脑组织匀浆和脑脊液（CSF）中脑啡肽酶的特异性酶活性。该检测使用了一种脑啡肽酶特异性抗体（之前用于市售ELISA试剂盒），在加入荧光肽底物（Mca-RPPGFSAFK（Dnp））之前，从脑匀浆和CSF中分离并固定NEP。这种荧光底物通常被多种酶裂解。我们已经表明，在没有免疫捕获阶段的情况下，即使在报道为对脑啡肽酶具有特异性的反应条件下-即在thiorphan存在下，在pH高于7时-荧光肽底物也不允许将脑匀浆和CSF中的脑啡肽酶活性与其他密切相关的酶的活性区分开。免疫捕获酶活性测定的特异性通过在thiorphan存在下脑匀浆和CSF中底物裂解的>80%抑制来证实。该测定允许高通量分析，并且关键的是，即使在测定粗组织匀浆或CSF时也确保高水平的酶特异性。(c)2007 Elsevier B. V.保留所有权利。
Neprilysin, a zinc-metalloendopeptidase, has important roles in the physiology and pathology of many diseases such as hypertension, cancer and Alzheimer's disease. We have developed an immunocapture assay to measure the specific enzyme activity of neprilysin in brain tissue homogenates and cerebrospinal fluid (CSF). The assay uses a neprilysin-specific antibody, previously used in a commercially available ELISA kit, to isolate and immobilise NEP from brain homogenates and CSF, prior to the addition of a fluorogenic peptide substrate (Mca-RPPGFSAFK(Dnp)). This fluorogenic substrate is ordinarily cleaved by multiple enzymes. We have shown that without the immunocapture phase, even under reaction conditions reported to be specific for neprilysin - i.e. in the presence of thiorphan, at pH above 7 - the fluorogenic peptide substrate does not allow neprilysin activity in brain homogenates and CSF to be discriminated from that of other closely related enzymes. The specificity of the immunocapture enzyme activity assay was confirmed by >80% inhibition of substrate cleavage in brain homogenates and CSF in the presence of thiorphan. The assay allows high-throughput analysis and, critically, also ensures a high level of enzyme specificity even when assaying crude tissue homogenates or CSF. (c) 2007 Elsevier B.V. All rights reserved.