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!

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.

July 2021 News

      You may have noticed that we recently updated our homepage, as our previous version became unstable for some unknown probably computer code related reason. We hope you like the newer version.
      We are glad to report that our recent paper, “TNFα increases tyrosine hydroxylase expression in human monocytes”, an early version of which was posted on the BioRΧiv server has been accepted for publication in NPJ Parkinson’s disease and can now be freely downloaded, from the journal here and also from Pubmed here. The Nature partner journals series, abbreviated NPJ, are a series of online-only, open access journals spun off by the journal Nature, one of the most prestigious and respected in science. This high impact journal is associated with the journal Parkinson’s Foundation and so provides a good venue to contact scientists and clinicians engaged in Parkinson’s disease research. The paper makes use of a sensitive and novel tyrosine hydroxylase ELISA developed using EnCor antibodies MCA-4H2 and RPCA-TH which we are now preparing for commercialization. For a write up of this work published on the University of Florida web site see here.
      We also release a novel goat polyclonal antibody to neurofilament NF-H GPCA-NF-H. This protein is heavily expressed in axons and is also released into blood and CSF following axonal compromise providing a useful surrogate marker of axonal damage and degeneration.
      Finally we continue to characterize the epitopes for our various monoclonal antibodies. An interesting recent finding it the mapping of one of our neurofilament NF-L monoclonals MCA-1B11. We showed that this binds to a highly conserved region of the dimeric α-helical coiled coil region of human NF-L. Much interest has been focused on the use of a neurofilament NF-L ELISA from Uman Diagnostics to monitor axonal loss following CNS compromise. The NF light Uman assay which utilizes two mouse monoclonal antibodies, clone 2.1 for NF-L detection and clone 47.3 for NF-L capture (see Norgren et al. 2002). These two clones are also known as UD1 and UD2 respectively. Interestingly our MCA-1B11 binds to the same epitope as the Uman detection reagent, UD1/clone 2.1. We are currently characterizing the epitope for the other Uman reagent and will shortly submit our findings on this to peer review. This work will lead to a better understanding of exactly how the Uman assay works and may lead to the development of novel second generation NF-L assays.

July 2020 News

      Like many other laboratories with expertise with antibody production we have focused some urgent attention on the SARS-Cov2 virus and the ACE2 protein which it uses to enter and infect human cells. The region of the virus spike or S-protein and the corresponding virus binding region on the ACE2 protein were recently characterized so we made recombinant forms of both regions in E. coli (Prot-r-SARS-CoV2-bd and Prot-r-ACE2-bd). We now have a panel of excellent mouse monoclonal antibodies to the ACE2 binding site of the SARS-CoV2 S-protein, and are marketing the first two of these, MCA-5G8 and MCA-2G1. We also made an affinity purified rabbit polyclonal antibody raised against the same immunogen, RPCA-SARS-CoV2-bd. These antibodies can be used to identify the SARS-Cov2 virus on western blots and by immunofluorescence and may also block the binding of the virus to ACE2. Of general interest to the world, the SARS-CoV2 S-protein ACE2 binding region turns out to be an excellent immunogen, so that it is likely an ideal target for vaccine development.
      We release new antibodies to β-synuclein, a mouse monoclonal and a rabbit polyclonal, MCA-6A10 and RPCA-SNCB. β-synuclein, like the closely related α-synuclein, is a major brain protein concentrated in synaptic regions. We also release new antibodies to Annexin A5 MCA-6A12 and RPCA-ANXA5. Annexin A5 is a Calcium and membrane associated protein which is upregulated when cells become apoptotic. We also release two new mouse monoclonals directed against PEA-15 MCA-4D2 and MCA-4D117. This protein was discovered as a “phosphoprotein enriched in astrocytes of 15kDa” (PEA-15) and independently as a “protein enriched in diabetes” (PED) and so is sometimes referred to as PED/PEA-15. PEA-15 is one of the large family of proteins containing a death effector domain (DED) and appears to have important functions in the regulation of cell growth, apoptosis and glucose metabolism. Finally we release a goat polyclonal antibody to myelin basic protein, GPCA-MBP, which can be used to identify and study myelin sheathes, oligodendrocytes and certain types of Schwann cell.

June 2020 News

      We add more goat antibodies to high value targets, namely to 2′-5′-cyclic nucleotide phosphodiesterase (CNP), GPCA-CNP, glial fibrillary acidic protein (GFAP) GPCA-GFAP, and mCherry GPCA-mCherry. Antibodies to CNP are excellent markers of myelin sheathes, GFAP antibodies label astrocytes, neural stem cells and a few other nervous system cells while mCherry is a widely used genetically encoded fluorescent tracer. Also another scientific paper performed using EnCor antibodies with the described assays run in the EnCor Lab, Holmström et al. 2020. This paper measures the levels of phosphorylated neurofilament heavy chain (pNF-H), ubiquitin C-terminal hydrolase 1 (UCHL1) and GFAP in the cerebrospinal fluid (CSF) and sera of recovering spinal cord injury patients and concludes that pNF-H and GFAP levels particularly in CSF may be useful for future monitoring of damage, secondary injury and response to treatment in these patients.

February 2020 News

      We release a new antibody to FOX3/NeuN, GPCA-FOX3, an excellent marker of neuronal nuclei and perikarya. The original NeuN antibody has been widely used to identify and count neurons since only these cells express this protein. The NeuN immunogen was found to correspond to the RNA binding protein FOX3, and our new antibody was made against a recombinant construct including the N-terminal 100 amino acids of human FOX3. The antibody was made in goat and so is very useful for double and triple label of mouse and other rodent species.