August 2022 News

      The Uman Diagnostics NF-LIGHT™ ELISA is a widely used ELISA type assay based on two mouse monoclonal antibodies to the neurofilament NF-L protein. One Uman antibody called variously UD1 or 2.1 is the detection reagent and the other, UD2, also known as 47.3, is the capture reagent (see Norgren et al. 2002). The same pair of antibodies are used in the more sensitive “Single Molecule Array” (Simoa™) assay marketed by Quanterix. These NF-L based assays have become very informative for the quantitation of axonal loss associated with a variety of CNS damage and disease states, see for example Barro et al. 2020 and Gaetani et al. 2019. We now have fully characterized the epitopes for both antibodies and have deposited the basic data on a preprint server see BioRχiv 2022.08.27.504533v1. The data in this report is currently undergoing peer review. These findings greatly increase understanding of what the Uman NF-LIGHT™ ELISA and the Quanterix Simoa™ assay are actually measuring and sets the stage for further NF-L assays with possibly improved properties.
      To our great surprise, neither Uman antibody stained sections of healthy CNS tissue with a typical NF-L pattern. However axons which were damaged as a result of experimental spinal cord injury in rats were strongly reactive with both Uman reagents. We hypothesized that the Uman epitopes were masked in assembled neurofilaments and made available to antibody binding by degeneration induced proteolysis. In agreement with this hypothesis we could make previously Uman negative control tissues strongly Uman positive by treatment with proteases. In addition fresh CNS tissues did not stain with Uman reagents while tissues left to sit at room temperature for 4 hours were strongly reactive. We also discovered that our antibodies to the C-terminal of NF-L, such as our rabbit polyclonal RPCA-NF-L-ct and mouse monoclonal MCA-DA2 stained neurofilaments in healthy processes but generally did not stain the degraded Uman positive NF-L positive material. On closer examination we determined that during injury induced degeneration processes positive for the C-terminal of NF-L became swollen and beaded. They apparently start to express the Uman epitopes and lose the NF-L C-terminal epitopes at about the same time. The final product is mostly diffuse and globular Uman positive degenerated axonal material. The Uman reagents and our NF-L C-terminal antibodies can therefore be used to positively identify both healthy, degenerating and degenerated processes.
      Based on these findings we made a novel panel of antibodies, both monoclonal and polyclonal, to the peptide containing both Uman epitopes. We have named these “DegenoTag™” reagents, as they specifically identify degenerated processes. These have the same properties of the Uman reagents as described above. We therefore have a panel of well characterized antibodies which can be used in the development of ELISA reagents and in immunocytochemical studies of normal, damaged and diseased CNS tissues. The first of these are MCA-6H63, MCA-1B11 and MCA-1D44. MCA-6H63 has an epitope a few amino acids N-terminal to that of UD2 while the epitope for MCA-1D44 is very similar to that of UD2. MCA-1B11 has an epitope similar to that of UD1. MCA-1D44 and MCA-6H63 share the interesting Uman property of only binding to degenerating and degenerated cells and processes, while MCA-1B11 also stains this degenerated material strongly but shows some reactivity with undamaged neurons and processes. We have also developed a rabbit polyclonal antibody RPCA-NF-L-Degen to the same region of NF-L which shares the property of only recognizing forms of NF-L expressed in degenerating processes.
      We publish another research report in collaboration with scientists at the University of Florida. This is “DAT and TH expression marks Human Parkinson’s Disease in Peripheral Immune Cells” by Gopinath et al. from the lab of Habibeh Khoshbouei in the journal “npj Parkinson’s Disease”. This article make use of our tyrosine hydroxylase (TH) antibodies to study blood cells in Parkinson’s disease patients. We also release an excellent and novel goat polyclonal antibody to tyrosine hydroxylase GPCA-TH.