Quality evaluation of monoclonal antibodies suitable for immunomagnetic purification of native tau protein

Tau protein plays a crucial role in the neuronal cytoskeleton stabilization. Under the pathological conditions it can be abnormally phosphorylated which leads to aggregation and formation of neurofibrillary tangles representing pathological hallmark of Alzheimer's disease (AD). For its association with neurodegenerative diseases tau protein is intensively studied in various diagnostic and therapeutic applications. Since there is no standard of tau protein involving essential post-translational modifications it is often necessary to purify it directly from cerebrospinal fluid (CSF) or blood of healthy or AD clinical signs exhibiting organism. Immunomagnetic purification based on the isolation of target protein using a specific antibody bound to magnetic carrier is the most effective tool for this purpose. High quality antibodies are the main prerequisite of successful purification, but many commercial antibodies does not comply with the challenging requirements for immunosorbent preparation. In this work, we compared 4 different anti-tau monoclonal antibodies currently available on the market (clones HT7, BT2, 8F10, 7E5). The evaluation criteria were set along the intended use for the preparation of specific magnetic immunosorbent subsequently applicable for native tau protein purification. We evaluated the characteristics declared by producers as specificity, purity and homogeneity. We also tested the binding affinity and IgG stability during the covalent immobilization to the surface of magnetic microparticles and during the immunoprecipitation of intact tau protein or tryptic tau fragments. Results are summarized and discussed here

Quality evaluation of monoclonal antibodies suitable for immunomagnetic purification of native tau protein

Tau protein plays a crucial role in the neuronal cytoskeleton stabilization. Under the pathological conditions, it can be abnormally phosphorylated which leads to the aggregation and formation of neurofibrillary tangles representing pathological hallmark of Alzheimer’s disease (AD). For its association with neurodegenerative diseases, tau protein is intensively studied in various diagnostic and therapeutic applications. Since there is no standard of tau protein involving essential post-translational modifications, it is often necessary to purify it directly from cerebrospinal fluid (CSF) or blood of healthy or AD clinical signs exhibiting organism. The immunomagnetic purification based on the isolation of the target protein using a specific antibody bound to a magnetic carrier is the most effective tool for this purpose. High quality antibodies are the main prerequisite of successful purification, but many commercial antibodies do not comply with the challenging requirements for the immunosorbent preparation. In this work, we compared four different anti-tau monoclonal antibodies currently available on the market (clones HT7, BT2, 8F10, 7E5). The evaluation criteria were set along the intended use for the preparation of specific magnetic immunosorbent subsequently applicable for the native tau protein purification. We evaluated the characteristics declared by producers as specificity, purity and homogeneity. We also tested the binding affinity and IgG stability during the covalent immobilization to the surface of magnetic microparticles and during the immunoprecipitation of intact tau protein or tryptic tau fragments. The results are summarized and discussed here

Magnetické částice s vázanými kinázami pro sekvenční in vitro fosforylace peptidů a proteinů

Post-translational modifications, including phosphorylation, greatly impact the physiological function of proteins, especially those that are natively unfolded and implicated in many neurodegenerative diseases. However, structural and functional studies of such proteins require fully defined phosphorylation, including those that are not physiological. Thus, the kinases ERK2 and GSK-3β were immobilized to various superparamagnetic beads with carboxylic, aldehyde, Ni2+, or Co3+ functional groups, with a view to efficiently phosphorylate peptides and proteins in vitro. Full phosphorylation of specific synthetic peptides confirmed that beads were successfully loaded with kinases. Remarkably, enzymes covalently immobilized on carboxylated SeraMag beads remained active upon reuse, with residual activity after 10 uses 99.5 ± 0.34% for GSK-3β and 36.2 ± 2.01% for ERK2. The beads were also used to sequentially phosphorylate recombinant tau, which in vivo is a biomarker of Alzheimer´s disease. Thus, a system consisting of two fully active kinases immobilized to magnetic beads is demonstrated for the first time. In comparison to soluble enzymes, the beads are easier to handle, reusable, and thus low-cost. Importantly, these beads are also convenient to remove from reactions to minimize contamination of phosphorylated products or to exchange with other kinases.Posttranslační modifikace včetně fosforylace významně ovlivňují fyziologickou funkci bílkovin, zvláště těch přirozeně nesbalených a zapojených do mnoha neurodegenerativních onemocnění. Nicméně strukturální a funkční studie takových proteinů vyžadují plně definovanou fosforylaci, včetně těch, které nejsou fyziologické. Proto byly kinázy ERK2 a GSK-3p imobilizovány na různé superparamagnetické částice s karboxylovými, aldehydovými, Ni2 + nebo Co3 + funkční skupiny, s cílem účinně fosforylovat peptidy a proteiny in vitro. Úplná fosforylace specifických syntetických peptidů potvrdila úspěšnou vazbu kináz. Enzymy kovalentně imobilizované na karboxylovaných částicích SeraMag zůstaly aktivní při opakovaném použití, přičemž po 10 použitích byla reziduální aktivita 99,5 ± 0,34% pro GSK-3β a 36,2 ± 2,01% pro ERK2. Částice byly také použity k postupné fosforylaci rekombinantního tau, který je v podmínkách in vivo biomarkerem Alzheimerovy choroby. Systém sestávající ze dvou plně aktivních kináz imobilizovaných na magnetických částicích je tak představen poprvé. Ve srovnání s rozpustnými enzymy jsou fosforylace s enzymy vázanými na částicích snadněji proveditelné, je možné je opakovaně použít a tedy s nízkými náklady. Důležité je, že tyto částice jsou také snadno odstranitelné z fosforylačních reakcí, a minimalizuje se tak kontaminace fosforylovaných produktů nebo se tak umožní výměna s jinými kinázami

N-Methyl-d-Aspartate Receptor – Nitric Oxide Synthase Pathway in the Cortex of Nogo-A-Deficient Rats in Relation to Brain Laterality and Schizophrenia

It has been suggested that Nogo-A, a myelin-associated protein, could play a role in the pathogenesis of schizophrenia and that Nogo-A-deficient rodents could serve as an animal model for schizophrenic symptoms. Since changes in brain laterality are typical of schizophrenia, we investigated whether Nogo-A-deficient rats showed any signs of disturbed asymmetry in cortical N-methyl-D-aspartate (NMDA) receptor–nitric oxide synthase (NOS) pathway, which is reported as dysfunctional in schizophrenia. In particular, we measured separately in the right and left hemisphere of young and old Nogo-A-deficient male rats the expression of NMDA receptor subunits (NR1, NR2A and NR2B in the frontal cortex) and activities of NOS isoforms (neuronal (nNOS), endothelial (eNOS) and inducible (iNOS) in the parietal cortex). In young controls, we observed right/left asymmetry of iNOS activity and three positive correlations (between NR1 in the left and NR2B laterality, between NR2B in the right and left sides, and between NR1 in the right side and nNOS laterality). In old controls, we found bilateral decreases in NR1, an increase in NR2B in the right side and two changes in correlations in the NR1–nNOS pathway. In young Nogo-A-deficient rats, we observed an increase in iNOS activity in the left hemisphere and two changes in correlations in NR1–nNOS and NR2A–eNOS, compared to young controls. Finally, we revealed in old Nogo-A-deficient animals, bilateral decreases in NR1 and one change in correlation between eNOS–iNOS, compared to old controls. Although some findings from schizophrenic brains did not manifest in Nogo-A-deficient rats (e.g., no alterations in NR2B), others did (e.g., alterations demonstrating accelerated ageing in young but not old animals, those occurring exclusively in the right hemisphere in young and old animals and those suggesting abnormal frontoparietal cortical interactions in young animals)

Hippocampal Spatial Position Evaluation on MRI for Research and Clinical Practice

<div><p>In clinical practice as well as in many volumetric studies we use different reorientations of the brain position towards x and y axis on the magnetic resonance imaging (MRI) scans. In order to find out whether it has an overall effect on the resulting 2D data, manual hippocampal area measurements and rotation variability of the brain (in two reoriented axes) and the skull were performed in 23 Alzheimer's disease patients and 31 healthy controls. After the MRI scanning, <i>native brain scans</i> (nat) were reoriented into the two different artificial planes (<i>anterior commissure – posterior commissure</i> axis (AC-PC) and <i>hippocampal horizontal long axis</i> (hipp)). Hippocampal area and temporal horn of the lateral ventricle was measured manually using freeware Image J program. We found that 1) hippocampal area of nat images is larger compared to hipp images, area of the nat images is equal to the AC-PC images and area of the hipp images is smaller compared to AC-PC images, 2) hippocampal area together with the area of the temporal horn for nat images is larger compared to hipp images, area of the hipp images is smaller compared to the AC-PC images and area of the nat images is smaller compared to the AC-PC images. The conclusion is that the measured area of the hippocampus in the native MRI is almost the same as the area of MRI reoriented only into the AC-PC axis. Therefore, when performing 2D area studies of the hippocampus or in the clinical practice we recommend usage of not-reoriented MRI images or to reorient them into the AC-PC axis. Surprising finding was that rotation of both AC-PC and hipp line towards x-axis among patients varies up to 35° and the same is true for the skull rotation so that it is not only a matter of the brain position.</p></div