pNF-H ASSAY SERVICE

We have developed a series of assays to the phosphorylated axonal form of the major neurofilament subunit, which we call pNF-H. This protein is one of the most major components of axons and is relatively easy to detect using antibody based assays as it contains unusual highly immunogenic repeated sequences and is resistant to proteases. This being the case, pNF-H is likely to be detectable following axonal damage or degeneration in biological fluids such as cerebrospinal fluid (CSF) and blood. The detection of pNF-H in these fluids is therefore a biomarker of axonal loss, which occurs rapidly following acute events such as traumatic brain and spinal cord injury and stroke. Axonal loss occurs slowly in neurodegenerative states such as Amyotrophic Lateral Sclerosis (ALS- a.k.a. Lou Gehrig’s disease), Multiple Sclerosis and secondary to more acute CNS challenges. The pNF-H assay we developed and assays developed by others have been used in many peer reviewed publications with the basic finding that low levels of pNF-H in CSF or blood predict a better patient or experimental animal outcome than higher levels, and that sudden release of pNF-H indicates acute CNS compromise.

Currently we have two pNF-H ELISA kits, one the original one described in our first paper which is based on an affinity purified chicken capture antibody and an affinity purified rabbit detection antibody (1). The second assay was described in a later paper and uses two mouse monoclonal antibodies (2). We have continued to develop pNF-H assays and now have available third generation assays in both the “classical” chromogenic ELISA format and the more sensitive Meso Scale Discovery (MSD) format. The MSD format uses a 96 well plate but detection is by electrochemiluminescense using a specialized Sector Imager, which is basically a highly sensitive camera which scans the 96 well plate. In this case we use a mouse monoclonal antibody for pNF-H capture and a tagged chicken antibody for detection.

We are also able to run any of the three pNF-H ELISA assays on a fee for service basis in the EnCor laboratory. For details of our pNF-H assays, including complete details of the assay validation, email assays@encorbio.com

References:

1. Shaw G, Yang C, Ellis R, Anderson K, Parker Mickle J, Scheff S, Pike B, Anderson DK and Howland DR. Hyperphosphorylated neurofilament NF-H is a serum biomarker of axonal injury. Biochem Biophys Res Commun. 336:1268-1277 (2005).

2. Boylan, K., Yang, C., Crook, J., Overstreet, K., Heckman, M., Wang, Y., Borchelt, D. and Shaw, G. 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 111:1182-1191 (2009).