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.
