December 2022 News
We release two new protein constructs, PROT-r-NF-L-Rct and PROT-r-NF-L-Stan. These constructs are designed to be used as protein standards for current neurofilament NF-L antibody based assays, such as the Uman/Quanterix NF-LIGHT™ and Quanterix Simoa™ assays. The two antibodies used in these assays both bind to the center of the “Coil II” region of α-helical “Rod” region of the NF-L molecule. The PROT-r-NF-L-Rct contains contains the entire Coil II region. The PROT-r-NF-L-Stan constructs contains the epitopes for both antibodies with little flanking sequence and is engineered to include 2 tryptophan residues. The result is a convenient low molecular weight protein standard which due to the high tryptophan content can be quantified spectrophotometrically more accurately than native or recombinant NF-L. Details of the mapping of the relevant NF-L assay antibodies and our generation of a novel panel of similar products are described in a recent BioRχiv publication, 10.1101/2022.08.27.504533v1.
We hired an excellent immunohistochemist to the great dismay of the University of Florida so we will be validating all our antibodies on formalin fixed paraffin embedded sections, in other words for immunohistochemistry (IHC). The harsh fixation and processing typically used with IHC heavily modifies immunogens and it is therefore expected to cause some otherwise useful antibodies to not work well. However certain other antibodies may recognize epitopes which are not damaged by IHC and so should work just fine. So we added to the “Additional Info” tag of the relevant product web pages images and details of positive IHC results. We now present data on many of our chicken antibodies; Specifically MAP-τ CPCA-Tau, neurofilament NF-H CPCA-NF-H, neurofilament NF-L CPCA-NF-L, tyrosine hydroxylase CPCA-TH, FOX3/NeuN CPCA-FOX3, FOX2 CPCA-FOX2, mCherry CPCA-mCherry, GFP CPCA-GFP, GFAP CPCA-GFAP, IBA1 CPCA-IBA1, MAP2A/B CPCA-MAP2, calbindin CPCA-Calb, myelin basic protein CPCA-MBP, CNPase CPCA-CNP, secretogogin CPCA-SCGN, α-synuclein CPCA-SNCA and visinin-like protein 1 CPCA-VLP1. We have also validated several goat antibodies for formalin fixed paraffin embedded IHC including our antibody to CNPase GPCA-CNP, FOX3/NeuN GPCA-FOX3, GFAP GPCA-GFAP, GFP GPCA-gfP,
MAP2 GPCA-MAP2, MBP GPCA-MBP, mCherry GPCA-mCherry, NF-H GPCA-NF-H and tyrosine hydroxylase GPCA-TH. We have also validated some of our mouse monoclonal antibodies on paraffin embedded IHC, such as DegenoTag™ NF-L antibody MCA-1B11, antibody to mCherry MCA-1C51,
Over the next few weeks we will validate all of our other chicken, mouse, rabbit and goat antibodies for IHC on human and rodent tissues, significantly enhancing their utility. We have already validated certain of our most widely used monoclonal antibodies such as MCA-1B7 which binds to FOX3/NeuN, an excellent marker of neurons, our rabbit polyclonal antibody to IBA1, RPCA-IBA1 an excellent marker of microglia and our monoclonal antibody to ubiquitin MCA-UBI-1, an excellent marker of Alzheimer neurofibrillary tangles and other pathological inclusions.
We usually go to the normally annual Society for Neuroscience Meeting, but missed 2020 and 2021 due to the pandemic. This year the meeting was in San Diego, November 11-16, and it was fun to see various people for the first time in three years. We were at booth 401 and handed out ~1,000 of our well known poster images for free, and they had all gone by the middle of the second day. We took along a few of our slides of brain sections and HeLa cell stained with various of antibodies giving 5 distinctly different fluorescence signals at 405nm, 488nm, 541nm, 645nm and 750nm. So on the brain sections it is possible to visualize on one section some combination of nuclei, neuronal cell bodies, neuronal dendrites, myelin sheaths, axons, astrocytes and microglia. In the HeLa cells you could see some combination of nucleus, mitochondria, microfilaments, intermediate filaments, microtubules, plasma membrane, nucleoli and nuclear lamina. It turned out that the various microscope companies loved these, as they had apparently all been looking for such samples to demonstrate how their various 2 photon, confocal, fluorescence etc. microscopes worked. So we are starting to market these as a new line of products. For no particular reason Dr. Shaw also brought along some Ramon-Y-Cajal images which he had burned into wood using a laser and tastefully framed. These turned out to be very popular also, and we sold all of them. So there is yet another potential line of products, watch this space!
September 2022 News
Our recent progress with the characterization of NF-L antibodies allow us to generate a novel and useful new reagent. This is our NF-L biomarker assay standard PROT-r-NF-L-Stan. This is a small recombinant protein which includes the epitopes for the two antibodies used in the NF-LIGHT™ and Quanterix Simoa™ NF-L biomarker assays. The recombinant construct is engineered to include tryptophan residues which allow accurate spectrophotometric quantitation of protein concentration by measuring optical density at a wavelength of 280nm. The generation of this product is based on the discoveries outlined in our recent BioRχiv article, see www.biorxiv.org/content/10.1101/2022.08.27.504533v1.
Our antibodies continue to be useful throughout the world! For example our mouse monoclonal antibody to adenylcyclase type III MCA-1A12, a convenient and key marker of neuronal cilia, was employed as part of a very high profile publication from the Janelia Research Campus, Howard Hughes Medical Institute, which provides evidence that neuronal cilia form functional synapses with serotonergic axons, see Cell 185:3390-3407 (2022). See a description of this work on the Janelia website here.
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 the Quanterix Simoa™ is 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 patient. We also release an excellent and novel goat polyclonal antibody to tyrosine hydroxylase GPCA-TH.
