CSF AND PLASMA NEUROFILAMENTS IN TRAUMATIC BRAIN INJURY

本提案的目的是开发急性神经元变性的实验室测试。这样的测试需要用于几种临床适应症，包括创伤性脑损伤（TBI）以及神经保护剂药物试验的分期患者。拟议的实验室测试将量化神经丝蛋白的水平。神经丝蛋白是主要定位于轴突腔室的神经元特异性蛋白。在正常情况下，神经丝蛋白不会从神经元中释放出来。然而，我们的初步Western blot研究表明，在神经损伤（TBI）患者后，脑脊液（CSF）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基NF-L， NF-M和NF-H的单克隆抗体（mab）。针对与NF-M和NF-H的n端区域具有70%同源性的重组NF-L，将建立单克隆抗体。识别NF-L、NF-M和NF-H的克隆将被鉴定，随后通过TBI和对照患者CSF的western blot筛选，以鉴定对CSF形式的NF-L、NF-M和NF-H具有免疫反应的克隆。确定的克隆将用于夹心ELISA开发。开发的神经丝ELISA将用于筛选TBI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。开发的神经丝酶联免疫吸附试验区分脑损伤患者脑脊液和血浆与对照患者的敏感性和特异性将被统计学确定。建议的商业应用一种在临床上有用的测试，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.