detection-disease-associated-synuclein-enhanced-elisa-brain

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

AJ Roboscreen GmbH

As an alternative method for challenge experiments in fish a sandwich-ELISA has been established for the detection of potentially protective antibody populations. Assay specificity depends on monoclonal antibodies (mabs) directed against different antigens of Aeromonas salmonicida, the causative agent of furunculosis. Comparable levels of salmon antibodies against LPS and the A-layer protein were recorded by ELISA and antigen binding assay. These studies, together with the first analysis of trout sera refer to the specificity and suitability of the ELISA-system for monitoring of individual antibody populations. A study of different mabs against the A-layer protein points to the possibility for detecting at least partially different antibody populations against this virulence factor.

[Development of an ELISA-system for the assessment of furunculosis vaccines]

The present study shows that a monoclonal antibody (MAb) directed to Aeromonas salmonicida glycerophospholipid:cholesterol acyltransferase (GCAT) is capable of protecting rainbow trout red blood cells from the cytotoxic effects of the enzyme in vitro.

Inhibition of hemolytic activity of Aeromonas salmonicida GCAT in rainbow trout red blood cells by a monoclonal antibody.

Rapid BSE tests are widely used diagnostics in veterinary medicine and more than 11 million tests are applied worldwide. The evaluation of new rapid BSE tests and the quality assurance of approved BSE tests pose a challenge owing to the natural scarcity of BSE-infected bovine brainstems and regional variations in prion titer. Transgenic mice expressing bovine prion protein (Tg4092) offer an alternative approach to these problems. To determine whether BSE-infected Tg4092 mouse brains could serve as a general standard for rapid BSE tests, we inoculated Tg4092 mice intracerebrally with BSE prions, harvested brains at defined time points post-infection and analyzed cerebral hemispheres with several approved rapid BSE tests. The results show that de novo formation of the disease-causing prion protein isoform, PrP(Sc), can be monitored during the course of infection. We demonstrate that BSE-infected Tg4092 mouse brains provide a renewable and controllable source of reference samples and suggest that such samples can generally be used for the evaluation and quality control of rapid BSE tests.

Transgenic mouse brains for the evaluation and quality control of BSE tests.

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that exerts a variety of physiological functions and is involved in various pathoprocesses. To characterize the role of tTG in disease, simple assays are necessary for detection. We developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) that combines a transamidation step with immunological detection to determine tTG. This assay is based on covalent binding of in vitro activated tTG to N,N'-dimethylated casein and subsequent detection of coupled tTG by specific immunoglobulin G (IgG) antibodies directed against tTG followed by binding of a secondary biotin-labeled antibody. The assay reaches a detection limit of 0.05ng of tTG/ml. Type 1 and 3 transglutaminases and factor XIII are not detected. By use of this assay, tTG in several cell lines and the stimulatory effect of retinoic acid on tTG expression could be demonstrated. The new assay represents a promising tool for the study of tTG in normal cell physiology and under pathological conditions.

Immunoassay of in vitro activated human tissue transglutaminase.

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes crosslinking of peptidic glutamine residues with primary amines via isopeptide bonds and hydrolysis of ATP or GTP. The enzyme exerts a variety of functions at the cellular and tissue levels that may be disturbed in disease. Its role in pathoprocesses is poorly understood. For investigation of the involvement of tTG in disease, sensitive and specific assays should be available. We have developed the first sandwich enzyme-linked immunosorbent assay (ELISA) based on two monoclonal antibodies (mabs) against human tTG. tTG is captured by mab 3C10 and detected by biotinylated mab 10F3. After incubation with peroxidase-conjugated streptavidin, bound tTG is visualized by peroxidase reaction applying a luminescence substrate. The detection limit was 40 pg/ml. The assay was highly reproducible. Recovery of spiked tTG in crude samples was greater than 92%. The enzyme could be detected in cellular lysates and tissue homogenates of humans. The effect of typical effectors (retinoic acid and interferon-γ) on tTG expression could be demonstrated. A low signal was also obtained in mice samples, suggesting cross-reactivity of the mabs with murine tTG. The new sandwich ELISA may be successfully applied for investigation of physiological functions of tTG and of disorders associated with inadequate tTG expression.

Quantification of human tissue transglutaminase by a luminescence sandwich enzyme-linked immunosorbent assay.

α-Synuclein is the major protein associated with Lewy body dementia, Parkinson's disease and multiple system atrophy. Since α-synuclein is present in the brain in physiological conditions as a presynaptic protein, it is crucial to characterize disease-associated modifications to develop an in vivo biomarker. With the aim to develop antibodies showing high specificity and sensitivity for disease-associated α-synuclein, synthetic peptides containing different amino acid sequences were used for immunization of mice. After generation of α-synuclein aggregates, ELISA and immunoblotting were used to test the specificity of antibodies. Tissue microarray sections originating from different human α-synucleinopathies were used to compare immunostaining with other, commercially available antibodies. Immunization of mice with the peptide TKEGVVHGVATVAE (amino acid 44-57 of α-synuclein) resulted in the generation of a monoclonal antibody (5G4), which was able to bind aggregated α-synuclein preparation in sandwich ELISA or coated on magnetic beads. 5G4 proved to be superior to other antibodies in comparative immunohistochemical studies by revealing more widespread and distinct α-synuclein pathology. Immunoblotting of human brain tissue revealed an additional band seen in dementia with Lewy bodies, whereas the band representing monomeric α-synuclein was very weak or lacking. In summary, the 5G4 antibody is most promising for re-evaluation of archival material and may offer new perspective for the development of in vivo diagnostic assays for detecting disease-associated α-synuclein in body fluids.

An antibody with high reactivity for disease-associated α-synuclein reveals extensive brain pathology.

Typical hallmarks of Alzheimer's disease (AD) are pathologic deposits in cortical and subcortical regions consisting of self-aggregated proteins such as amyloid-beta (Aβ) or tau. Tissue transglutaminase (tTG) catalyses calcium-dependent cross-linking between proteins (transamidation) resulting in protease-resistant isopeptide bonds. Because of this ability, tTG was suspected to participate in AD pathogenesis. Aβ and tau can be cross-linked by tTG in vitro. In AD neocortex, messenger RNA expression of tTG is increased. However, data on transamidation in AD specimens--activity of not only tTG but also other transglutaminases--are contradictory. The aim of our study was to investigate if tTG is involved in AD development and may be useful as biomarker for AD. We studied human brain samples for tTG concentration, tTG localization, and transamidation activity and cerebrospinal fluid (CSF) for tTG content by novel sensitive and highly specific methods. Neither tTG concentration nor transamidation was increased in AD brain homogenates. Immunohistologically, we found no colocalization of tTG in neocortex sections with tau or Aβ deposits but with blood vessels. Only in rare cases, tTG was detectable in CSF samples. This could be attributed to liberation from erythrocytes. Our data contradict the view that tTG is a potential biochemical marker for AD.

Tissue transglutaminase is not a biochemical marker for Alzheimer's disease.

Tissue transglutaminase in Alzheimer's disease - facts and fiction: a reply to "Tissue transglutaminase is a biochemical marker for Alzheimer's disease".

Dementia with Lewy bodies (DLB), Parkinson's disease (PD) and multiple system atrophy are characterized by the deposition of disease-associated α-synuclein. In the present study we 1) examined the molecular specificity of the novel anti-α-synuclein 5G4 antibody; 2) evaluated immunoreactivity patterns and their correlation in human brain tissue with micro- and astrogliosis in 57 cases with PD or DLB; and 3) performed a systematic immunoelectron microscopical mapping of subcellular localizations. 5G4 strongly binds to the high molecular weight fraction of β-sheet rich oligomers, while no binding to primarily disordered oligomers or monomers was observed. We show novel localizations of disease-associated α-synuclein including perivascular macrophages, ependyma and cranial nerves. α-Synuclein immunoreactive neuropil dots and thin threads associate more with glial reaction than Lewy bodies alone. Astrocytic α-synuclein is an important component of the pathology. Furthermore, we document ultrastructurally the pathway of processing of disease-associated α-synuclein within neurons and astroglial cells. Interaction of mitochondria and disease-associated α-synuclein plays a key role in the molecular-structural cytopathogenesis of disorders with Lewy bodies. We conclude that 1) the 5G4 antibody has strong selectivity for β-sheet rich α-synuclein oligomers; 2) Lewy bodies themselves are not the most relevant morphological substrate that evokes tissue lesioning; 3) both neurons and astrocytes internalize disease-associated α-synuclein in the human brain, suggesting prion-like cell-to-cell spread of α-synuclein by uptake from surrounding structures, as shown previously in experimental observations.

Intracellular processing of disease-associated α-synuclein in the human brain suggests prion-like cell-to-cell spread.

With the aim to evaluate the significance and reliability of detecting disease-specific α-synuclein in the cerebrospinal fluid (CSF) we developed an ELISA and bead-assay. We used a commercial antibody (5G4) that does not bind to the physiological monomeric form of α-synuclein, but is highly specific for the disease-associated forms, including high molecular weight fraction of β-sheet rich oligomers. We applied both tests in CSF from a series of neuropathologically confirmed α-synucleinopathy cases, including Parkinson' disease dementia (PDD) and dementia with Lewy bodies (DLB) (n = 7), as well as Alzheimer' disease (n = 6), and control patients without neurodegenerative pathologies (n = 9). Disease-specific α-synuclein was detectable in the CSF in a subset of patients with α-synuclein pathology in the brain. When combined with the analysis of total α-synuclein, the bead-assay for disease-specific α-synuclein was highly specific for PDD/DLB. Detection of disease-associated αsynuclein combined with the total levels of α-synuclein is a promising tool for the in-vivo diagnosis of α-synucleinopathies, including PDD and LBD.

Detection of disease-associated α-synuclein in the cerebrospinal fluid: a feasibility study.

Defects in intracellular transport are implicated in the pathogenesis of Alzheimer's disease (AD). Hook proteins are a family of cytoplasmic linker proteins that participate in endosomal transport. In this study we show that Hook1 and Hook3 are expressed in neurons while Hook2 is predominantly expressed in astrocytes. Furthermore, Hook proteins are associated with pathological hallmarks in AD; Hook1 and Hook3 are localized to tau aggregates and Hook2 to glial components within amyloid plaques. Additionally, the expression of Hook3 is reduced in AD. Modelling of Hook3 deficiency in cultured cells leads to slowing of endosomal transport and increases β-amyloid production. We propose that Hook3 plays a role in pathogenic events exacerbating AD.

Ingolf Lachmann

AJ Roboscreen GmbH

Detection of Disease-associated α-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated α-synuclein