Comparison of two ELISA methods for measuring levels of the phosphorylated neurofilament heavy chain (original) (raw)
Related papers
Hyperphosphorylated neurofilament NF-H is a serum biomarker of axonal injury
Biochemical and Biophysical Research Communications, 2005
Several lines of reasoning suggest that the phosphorylated axonal form of the neurofilament subunit NF-H is likely to be released from damaged and diseased neurons in significant amounts. Detection of this protein in serum or CSF might therefore provide information about the presence and degree of neuronal loss. We therefore developed a sensitive NF-H ELISA capable of detecting picogram quantities of phosphorylated NF-H (pNF-H). This assay showed that soluble pNF-H immunoreactivity is readily detectable in the sera of adult rats following various types of experimental spinal cord injury (SCI) and traumatic brain injury (TBI), but is undetectable in the sera of normal animals. Here we describe details of the time course and extent of serum pNF-H expression following experimental SCI and TBI. Following SCI, serum pNF-H showed an initial peak of expression at 16 h and a second, usually larger, peak at 3 days. Following TBI, lower levels of serum pNF-H were detected with a peak at 2 days post-injury. We also show that the higher levels of pNF-H released from injured spinal cord as compared to brain are in line with the $20-fold higher levels of pNF-H present in spinal cord. These findings suggest that serum levels of pNF-H immunoreactivity may be used to conveniently monitor neuronal damage and degeneration in experimental and presumably clinical situations.
A specific ELISA for measuring neurofilament heavy chain phosphoforms
Journal of Immunological Methods, 2003
Neurofilaments (Nf) are the major constitutents of the axoskeleton and body fluid Nf levels are an important tool for estimating axonal degeneration in vivo. This paper presents a new sandwich ELISA allowing quantification of the NfH SMI35 phosphoform from CSF, brain tissue and cell culture homogenates. The sensitivity of the NfH SMI35 ELISA is 0.2 ng/ml with a recovery of 119% and a mean within-and between-batch precision of 10.6% and 23%, respectively. CSF NfH SMI35 was stable at 4 jC, is not influenced by freeze -thaw cycles, and proteolysis present at room temperature could be prevented by adding protease inhibitors. Aggregate formation was observed for HPLC-purified bovine NfH and could be resolved by sonication.
Neurofilaments as biomarkers in neurological disorders
Nature Reviews Neurology, 2018
Please check that you are happy with how it reads and that no crucial information has been removed.] Neuroaxonal damage is the pathological substrate of permanent disability in various neurological disorders. Reliable quantification and longitudinal follow-up of such damage is important for assessing disease activity, monitoring treatment responses, facilitating treatment development and prognostic purposes. The neurofilament proteins have promise in this context because their levels rise upon neuroaxonal damage not only in the CSF, but also in blood, and they indicate neuroaxonal injury independent of causal pathways. First-generation (immunoblot) and secondgeneration (enzyme-linked immunosorbent assay) neurofilament assays were of limited sensitivity. Third-generation (electrochemiluminescence) and especially fourth-generation (single molecule array) assays enable reliable measurement of neurofilaments throughout the range of concentrations found in blood samples. This technological advancement has paved the way to investigate neurofilaments in a range of neurological disorders. Here, we review what is known about the structure and function of neurofilaments, discuss analytical aspects and knowledge of age-dependent normal ranges of neurofilaments and provide a comprehensive overview of studies on neurofilament light as a marker for axonal injury in different neurological disorders, including multiple sclerosis, neurodegenerative dementia, stroke, traumatic brain injury, amyotrophic lateral sclerosis and Parkinson disease. We also consider work needed to explore the value of this axonal damage marker in managing neurological diseases in daily practice.
Journal of Neurotrauma, 2008
The detection of neuron-specific proteins in blood might allow quantification of the degree of neuropathology in experimental and clinical contexts. We have been studying a novel blood biomarker of axonal injury, the heavily phosphorylated axonal form of the high molecular weight neurofilament subunit NF-H (pNF-H). We hypothesized that this protein would be released from damaged and degenerating neurons following experimental traumatic brain injury (TBI) in amounts large enough to allow its detection in blood and that the levels detected would reflect the degree of injury severity. An enzyme-linked immunosorbent assay (ELISA) capture assay capable of detecting nanogram amounts of pNF-H was used to test blood of rats subjected to experimental TBI using a controlled cortical impact (CCI) device. Animals were subjected to a mild (1.0 mm), moderate (1.5 mm), or severe (2.0 mm) cortical contusion, and blood samples were taken at defined times postinjury. The assay detected the presence of pNF-H as early as 6 h post-injury; levels peaked at 24-48 h, and then slowly decreased to baseline over several days post-injury. No signal above baseline was detectable in control animals. Analysis of variance (ANOVA) showed a significant effect of lesion severity, and post hoc analysis revealed that animals given a moderate and severe contusion showed higher levels of blood pNF-H than controls. In addition, the peak levels of pNF-H detected at both 24 and 48 h post-injury correlated with the degree of injury as determined by volumetric analysis of spared cortical tissue. Relative amounts of pNF-H were also determined in different areas of the central nervous system (CNS) and were found to be highest in regions containing large-diameter axons, including spinal cord and brainstem, and lowest in the cerebral cortex and hippocampus. These findings suggest that the measurement of blood levels of pNF-H is a convenient method for assessing neuropathology following TBI.
A multi-center study of neurofilament assay reliability and inter-laboratory variability
Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 2020
Objectives: Significantly elevated levels of neurofilament light chain (NfL) and phosphorylated neurofilament heavy chain (pNfH) have been described in the blood and cerebrospinal fluid (CSF) of amyotrophic lateral sclerosis (ALS) patients. The aim of this study was to evaluate the analytical performance of different neurofilament assays in a round robin with 10 centers across Europe/U.S. Methods: Serum, plasma and CSF samples from a group of five ALS and five neurological control patients were distributed across 10 international specialist neurochemical laboratories for analysis by a range of commercial and in-house neurofilament assays. The performance of all assays was evaluated for their ability to differentiate between the groups. The inter-assay coefficient of variation was calculated where appropriate from sample measurements performed across multiple laboratories using the same assay. Results: All assays could differentiate ALS patients from controls in CSF. Inter-assay coefficient of variation of analytical platforms performed across multiple laboratories varied between 6.5% and 41.9%. Conclusions: This study is encouraging for the growing momentum toward integration of neurofilament measurement into the specialized ALS clinic. It demonstrates the importance of 'round robin' studies necessary to ensure the analytical quality required for translation to the routine clinical setting. A standardized neurofilament probe is needed which can be used as international benchmark for analytical performance in ALS.
CSF phosphorylated neurofilament subunit NF-H (pNF-H) levels are biomarkers of Spinal Cord Injury
Romanian Neurosurgery, 2013
Several studies showed that the phosphorylated form of the neurofilament subunit NF-H (pNF-H) are related to neuronal injuries and its detection provide information about the presence and degree of neuronal loss. Neurofilaments are three subunits, namely NF-L, NF-M and NF-H. The phosphorylated neurofilament subunit NF-H (pNF-H) is present into serum and CSF in significant amounts following neuronal injury and may be detected. The pNF-H can be a biomarker of the neuronal injuries and its detection allows the monitoring neuronal pathology and may provide diagnosis and prognosis in humans. We are interested in pNF-H as biomarker of neuronal injury in spinal cord injury and we used a pNF-H ELISA test capable of detecting the levels of phosphorylated NF-H (pNF-H) to patients with spinal cord injury. We studied the pNF-H levels in CSF in two patients with spinal cord injury (SCI) and for normal values of pNF-H we determined the CSF pNF-H level from individuals without neurological damage....