CSF AND PLASMA NEUROFILAMENTS IN TRAUMATIC BRAIN INJURY

这项提议的目标是开发一种针对急性神经元变性的实验室测试。这样的测试对于包括创伤性脑损伤(TBI)在内的几个临床指征以及神经保护剂药物试验的分期患者来说都是必要的。拟议的实验室测试将量化神经丝蛋白的水平。神经丝蛋白是一种神经元特异性蛋白，主要定位于轴突室。在正常情况下，神经丝蛋白不会从神经元中释放。然而，我们的初步Western印迹研究表明，神经元损伤(TBI)后，脑脊液(CSF)中神经丝蛋白水平升高，而对照组脑脊液中未检测到神经丝蛋白水平。拟议的研究将产生识别神经丝亚单位NF-L、NF-M和NF-H的单抗(MAb)。针对与核因子-M和核因子-H的N-末端有70%同源性的重组核因子-L，将产生单抗。将鉴定识别NF-L、NF-M和NF-H的克隆，并随后通过与脑损伤和对照患者脑脊液的免疫印迹进行筛选，以鉴定与脑脊液形式的NF-L、NF-M和NF-H免疫反应阳性的克隆。经鉴定的克隆将用于夹心ELISA的开发。所建立的神经丝ELISA将用于筛选脑外伤患者(N=30)和对照组(N=67，脱髓鞘疾病、脑积水、背痛和偏头痛)的脑脊液和血浆。所建立的神经丝ELISA法区分脑外伤患者和对照组脑脊液和血浆的敏感性和特异性将在统计学上确定。拟议的商业应用一种临床上有用的测试，以帮助诊断头部损伤。

The objective of this proposal is to develop a laboratory test for acute neuronal degeneration. Such a test is needed for several clinical indications including traumatic brain injury (TBI) as well as, staging patients for drug trials of neuroprotective agents. The proposed laboratory test will quantify levels of neurofilament proteins. Neurofilament proteins are neuron- specific proteins localized primarily in the axonal compartment. Under normal conditions neurofilament proteins are not released from neurons. However, our preliminary Western blot studies indicated that after neuronal injury (TBI patients) cerebrospinal fluid (CSF) neurofilament protein levels were elevated while neurofilament levels in control CSF were not detectable. The proposed studies will raise monoclonal antibodies (Mabs) that recognize the neurofilament subunits NF-L, NF-M and NF-H. Mabs will be raised against recombinant NF-L that demonstrates a 70 percent homology with the N-terminal region of NF-M and NF-H. Clones recognizing NF-L, NF-M and NF-H will be identified and subsequently screened by western blot against TBI and control patient CSF to identify clones immunoreactive with the CSF form of NF-L, NF-M and NF-H. Identified clones will be employed for sandwich ELISA development. The developed neurofilament ELISA will be used to screen CSF and plasma from TBI patients (N=30) and controls (N=67, demyelinating disease, hydrocephalus, back pain and migraine). The sensitivity and specificity of the developed neurofilament ELISA to discriminate TBI patient CSF and plasma from control patients will be statistically determined. PROPOSED COMMERICAL APPLICATION A clinically useful test to aid in the diagnosis of head injury.