Research paperAn enzyme immunoassay to quantify neurofilament light chain in cerebrospinal fluid
Introduction
Neurofilament (NF) proteins are highly phosphorylated proteins and represent, beside microtubules, a major element of the neuronal cytoskeleton. Neurofilaments are predominantly localized in axons and are composed of three different proteins with molecular weights of 68 kDa (NF-light chain, NF-L), 160 kDa (NF medium chain, NF-M) and 200 kDa (NF high chain, NF-H), respectively. They play an important role in neuronal structure and function and it has been suggested that neurofilaments control the diameter of the axons in motor neurons. Of the three subunits, NF-L is the only neurofilament protein that is capable of organising filaments by itself (Carpenter and Ip, 1996).
NF-L can be detected in cerebrospinal fluid (CSF) and its concentration may reflect the degree of compromised axonal function or axonal degeneration. CSF NF-L levels correlate with increasing degrees to white matter changes in the brain (Sjogren et al., 2001a, Tullberg et al., 2002). High concentrations of NF-L have been observed in the CSF of patients with amyotrophic lateral sclerosis, in particular, in patients with upper motor neuron disease (Rosengren et al., 1996), in patients with cerebral vaculitis (Nylen et al., 2002), those with normal pressure hydrocephalus (Tullberg et al., 1998) and those with active relapsing remitting multiple sclerosis (Lycke et al., 1998). CSF NF-L levels were particularly elevated in patients with frontotemporal dementia in comparison to early-onset Alzheimer's disease (Sjogren et al., 2000). Within a group of patients with neurodegenerative movement disorders high levels of NF-L have been observed in patients with multiple system atrophy or progressive supranuclear palsy relative to Parkinson's disease (Holmberg et al., 1998).
The analysis of NF-L in CSF may thus be helpful in the differential diagnosis of several neurological disorders. However, widespread application of such analysis has been hampered by the lack of available methods. Rosengren and colleagues described an ELISA to quantify CSF NF-L using in-house antibodies (Rosengren et al., 1996). The aim of the present study was to develop an assay based on commercially available antibodies and antigen to measure NF-L in CSF and to investigate its potential value as a marker of axonal damage and neuronal degeneration in conditions of severe brain damage, such as subarachnoid hemorrhage, traumatic brain injury and cerebrovascular accidents.
Section snippets
Patients
All CSF samples were obtained by lumbar puncture (between 1997 and 2004) performed as part of the routine diagnostic procedures in the Department of Neurology, University Medical Center Nijmegen. The CSF was centrifuged and aliquots of each sample were immediately frozen at −80 °C until analysis.
The control group (n=110) consisted of patients who underwent a lumbar puncture as part of the diagnostic process but who, after extensive examination, turned out not to suffer from a neurological
Results
Fig. 1 shows a typical calibration curve for NF-L. The goodness of fit was 97.4%. The detection limit of the assay was calculated as the mean plus three standard deviations of the zero standard signal from 40 measurements and was determined to be 5 ng/l.
Discussion
In the present study we have described a sensitive and reproducible immuno-assay to quantify NF-L in CSF, based on commercially available antibodies and antigen, with a detection limit of 5 ng/l. Sensitivity was achieved using two overnight incubation steps at 4 °C with calibrator and sample and substrate, respectively, and using a combination of two monoclonal detection antibodies which increased sensitivity relative to a single antibody. In comparison with the results of Rosengren et al. (1996
Acknowledgement
We thank H.P.M. de Reus and R. Claessens-Linskens and other technicians of the Laboratory of Pediatrics and Neurology for their technical assistance. This study was supported by the Hersenstichting Nederland (8F00(2).31), Internationaal Parkinson Fonds and Senter (Ministry of Economical Affairs, The Netherlands).
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