Product name | ELISA pNF-H version 2 |
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Description | ELISA kit for the detection of pNF-H in plasma, serum, CSF and tissue extracts. |
Reference Code | ELISA pNF-H V2 |
HGNC name | NEFH |
Molecular weight | ~210kDa by SDS-PAGE, real MW ~110kDa |
Immunogen | Phosphorylated axonal NF-H (pNF-H) |
Isotype | Mouse monoclonal IgG capture and mouse monoclonal IgG detect |
Species Reactivity | Human, rat, mouse, horse, cow, pig, dog |
Applications | ELISA of plasma, serum, CSF and tissue extracts. |
Storage instructions | Shipped on ice. Store at 4°C. |
pNF-H ELISA Version 2
Cat# ELISA-pNF-H-V2
$400.00 – $1,800.00
Neurofilament subunit NF-H is one of the three so-called neurofilament triplet proteins which are major structural components of neurons. These proteins are particularly heavily concentrated in the axons of large projection neurons, where they clearly occupy the majority of the cytoplasm. The NF-H protein has an unusual C-terminal region which includes dozens of peptides based on the sequence lysine-serine-proline (KSP in the single letter amino acid code). In axons these KSP sites are heavily phosphorylated on the serine residues, while the form of NF-H in dendrites and perikarya is not normally phosphorylated (1,2). In consequence the KSP phosphorylated form of NF-H, usually referred to as pNF-H, is an axon specific form.
We have developed sensitive ELISA kits to accurately quantify levels of the pNF-H protein. The protein is abundant and resistant to proteases so that, after axonal injury or degeneration, pNF-H can be readily detected with an ELISA such as this one. A variety of serious damage and disease states are associated with axonal compromise, such as amyotrophic lateral sclerosis, multiple sclerosis, traumatic brain injury and traumatic spinal cord injury. A growing list of peer reviewed publications make use of this and similar assays to monitor ongoing axonal loss associated with these and a variety of CNS damage and disease states. The first version of the assay utilized two affinity purified polyclonal antibodies, a chicken for antigen capture and a rabbit for detection, and has been described in peer reviewed publications (3,4). This assay is marketed by EnCor and several other vendors. We also developed and market this second generation pNF-H assay using two mouse monoclonal antibodies, although the first generation assay is also available, see ELISA-pNF-H-V1. This second generation assay has also been described in a peer reviewed publication (5). The manual for this kit can be downloaded here.
Neurofilament subunit NF-H is one of the three so-called neurofilament triplet proteins which are major structural components of neurons. These proteins are particularly heavily concentrated in the axons of large projection neurons, where they clearly occupy the majority of the cytoplasm. The NF-H molecule has a highly unusual protein sequence which includes a highly repetitive region made up of slight variants of the 6-8 amino acid sequences based on the peptide lysine-serine-proline (KSP in the single letter code). There are about 50 of these tandemly repeated peptides in known mammalian NF-H sequences, the exact number being a little variable in different species. Interestingly, the serine residues are not phosphorylated in dendritic and perikaryal neurofilaments but are heavily phosphorylated in axonal neurofilaments, so that antibodies which recognize the KSP phosphorylated from NF-H, which we refer to as pNF-H, are excellent markers of axons (1,2). We have developed a sensitive ELISA kit to accurately quantify the level of expression of this protein in a variety of experimental situations. Since NF-H is expressed later in development, and becomes heavily phosphorylated later than the other two triplet proteins this kit can be used to monitor neuronal maturation. Neurofilament expression is up regulated in a variety of damage and disease states, and this kit can be used to accurately quantify this up regulation. We have also recently found that this kit can detect pNF-H in the sera of animals with spinal cord and brain lesions, suggesting the exciting possibility that pNF-H is a useful biomarker of neuronal and more specifically axonal injury or degeneration (3). Further studies show that this assay can detect informative levels of pNF-H in both the CSF and blood of humans suffering from a variety of neurological disorders (4,5,6). In fact, there are several reasons to think that pNF-H might be an unusually useful and informative biomarker of axonal injury and degeneration. Firstly, pNF-H is an abundant component of axons as noted above, so it should be released in relatively large quantity following axonal damage and degeneration. Secondly, it is unusually resistant to proteolysis, apparently partly due to the heavy phosphorylation (7,8), meaning that it is likely to be relatively long lived on release into CSF and blood, aiding its detection. Thirdly, the unusual multiepitope nature of the phosphorylation sites coupled with their great immunogenicity means that pNF-H can be captured and detected with unusual avidity by appropriate immunoreagents. Finally, pNF-H detection reflects axonal injury and much recent evidence suggests that a variety of damage and disease states can be regarded as the result of axonal injury (e.g. 9,10). The unique geometry of neurons renders the axons particularly sensitive to mechanical injury or local loss of oxygen or metabolic substrates, and our assay can determine how much of this axonal injury has occurred. This kit was recently used to show that pNF-H levels were greatly elevated in the CSF of patients with subacute sclerosing panencephalitis, and that the levels detected correlated well with the state of progression of the disease (11).
To develop this kit we developed two mouse monoclonal antibodies specifically for this purpose. The capture antibody is an unusually high affinity and specificity antibody and is therefore ideal for antigen capture. To detect the binding of pNF-H detection antibody we coupled it covalently to HRP.
The kit contains one standard ELISA 96 well plate coated with the capture antibody which has been blocked and so is ready to use. The plate can be separated into 8 12 well strips. The kit includes a pure pNF-H standard, the detection antibody coupled to HRP, p-Nitrophenol phosphatase substrate and buffer concentrates. We also include detailed instructions.
Name: | ELISA kit for detection of pNF-H |
Immunogen: | phosphorylated axonal form of NF-H from bovine spinal cord |
HGNC Name: | NEFH |
UniProt: | P12036 (human) |
Molecular Weight: | ~220kDa by SDS-PAGE, real molecular weight ~110kDa |
Host: | NA |
Isotype: | |
Species Cross-Reactivity: | Human, Rat, Mouse, Pig, Cow |
RRID: | Pending |
Format: | 96 Well ELISA |
Applications: | CSF and blood pNF-H detection |
Recommended Dilutions: | CSF or blood samples from animals or individuals with CNS injury or degeneration |
Storage: | Store at 4°C |
1. Sternberger LA, Sternberger NH. Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ. PNAS 80:6126-6130 (1983).
2. Lee VM et al. Identification of the major multiphosphorylation site in mammalian neurofilaments. PNAS 85:1998-2002 (1998).
3. Shaw G et al. Hyperphosphorylated neurofilament NF-H is a serum biomarker of axonal injury. Biochem. Biophys. Res. Commun. 336:1268-1277 (2005).
4. Petzold, A. and Shaw, G. Comparison of two ELISA methods for measuring levels of the phosphorylated neurofilament heavy chain. J. Immunol. Methods 319:34-40 (2007).
5. Boylan K et al. Phosphorylated neurofilament heavy subunit (pNF-H) in peripheral blood and CSF as a potential prognostic biomarker in amyotrophic lateral sclerosis. J. Neurochem. 111:1182-91 (2009).
6. Shaw G. The use and potential of pNF-H as a general blood biomarker of axonal loss: an immediate application for CNS injury. Chapter 21 in book “Brain Neurotrauma: Molecular, Neuropsychological, and Rehabilitation Aspects” Edited by Firas Kobeissy, CMC Press, 289-300 (2015).
7. Siman, R. et al. Biomarker evidence for mild central nervous system injury after surgically-induced circulation arrest. Brain Res. 1213:1-11 (2008).
8. Lewis, S. et al. Detection of phosphorylated NF-H in the cerebrospinal fluid and blood of aneurysmal subarachnoid hemorrhage patients. J Cereb Blood Flow Metab. Epub Mar 5 (2008).
9. Schlaepfer WW, Lee C, Lee VM, and Zimmerman UJ. An immunoblot study of neurofilament degradation in situ and during calcium-activated proteolysis, J. Neurochem. 44:502–9 (1985).
10. Pant HC. Dephosphorylation of neurofilament proteins enhances their susceptibility to degradation by calpain.
Biochem. J. 256:665-8 (1988).
11. Stys PK. General mechanism of axonal damage and its prevention. J. Neurol. Sci. 233:3-13 (2005).
12. Buki A and Povlishock JT. All roads lead to disconnection? – Traumatic axonal injury revisited.
Acta Neurochir. (Wien). 148:181-93 (2005).
13. Boylan KB. et al. Immunoreactivity of the phosphorylated axonal neurofilament H subunit (pNF-H) in blood of ALS model rodents and ALS patients: evaluation of blood pNF-H as a potential ALS biomarker. J. Neurochem. 111:1182-91 (2009).
14. Matsushige T, et al. CSF neurofilament and soluble TNF receptor 1 levels in subacute sclerosing panencephalitis. J. Neuroimmunol. 205:155-9 (2008).
15. Pasol et al. Phosphorylated Neurofilament Heavy Chain Correlations to Visual Function, Optical Coherence Tomography, and Treatment. Mult. Scler. Int. 542691 (2010).
16. Toedebusch CM. et al. Cerebrospinal Fluid Levels of Phosphorylated Neurofilament Heavy as a Diagnostic Marker of Canine Degenerative Myelopathy.
J. Vet. Intern. Med. 31:513–20 (2017).
17. Mashita et al. Combination of serum phosphorylated neurofilament heavy subunit and hyperintensity of intramedullary T2W on magnetic resonance imaging provides better prognostic value of canine thoracolumbar intervertebral disc herniation. J. Vet. Med. Sci. 77:433-8 (2015).
18. Boylan KB. et al. Phosphorylated neurofilament heavy subunit (pNF-H) in peripheral blood and CSF as a potential prognostic biomarker in amyotrophic lateral sclerosis. J. Neurol. Neurosurg. Psychiatry. 84:467-72 (2013).
19. Gresle MM. et al. Serum phosphorylated neurofilament-heavy chain levels in multiple sclerosis patients.
J. Neurol. Neurosurg. Psychiatry. 85:1209-13 (2014).
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Contact info
EnCor Biotechnology Inc.
4949 SW 41st Boulevard, Ste 40
Gainesville
Florida 32608 USA
Phone: (352) 372 7022
Fax: (352) 372 7066
E-mail: admin@encorbio.com