Jef Boons

Monoclonal antibodies with selective specificity for Alzheimer Tau are directed against phosphatase-sensitive epitopes.

SummaryA modified form of the microtubule-associated protein Tau is the major component of the paired helical filaments (PHF) found in Alzheimers disease. The characterization of these posttranslational Tau modifications is hindered by the lack of sufficient PHF-Tau-specific markers. Here we describe several monoclonal antibodies, prepared by immunization with PHF, two of which showed a selective specificity for PHF-Tau without cross-reactivity with normal Tau. Epitope recognition by these two monoclonals was sensitive to alkaline phosphatase treatment. In Western blotting these monoclonal antibodies reacted specifically with the abnormally phosphorylated epitopes on Alzheimers disease-associated PHF-Tau. One of the new antibodies can be used for the construction of a sandwich enzyme-linked immunosorbent assay for the specific detection of PHF-Tau without cross-reactivity to normal Tau proteins.

A prospective study of CSF markers in 250 patients with possible Creutzfeldt–Jakob disease

Objective: To investigate various cerebrospinal fluid (CSF) markers that could assist in the clinical diagnosis of Creutzfeldt–Jakob disease (CJD). Methods: CSF samples were analysed for the presence of 14-3-3 protein, microtubule associated protein tau, and β amyloid in 250 patients with possible CJD. Densitometric analysis was used to quantify the level of 14-3-3 in all patients. Results: Analysis of the clinical data showed that cerebellar signs or myoclonus combined with progressive dementia were the main features leading to a clinical suspicion of CJD. While 14-3-3 detection had a sensitivity of 100% and a specificity of 92%, tau determination using a threshold of 1300 pg/ml had a sensitivity of 87% and a specificity of 97%. If the protocol for the analysis of 14-3-3 was modified (using densitometric analysis) a higher specificity (97%) could be obtained, but with a lower sensitivity (96%). Maximum sensitivity, specificity, and positive predictive value were obtained with a combination of 14-3-3 and β amyloid determinations. The concentrations of 14-3-3 and tau in the CSF were reduced in CJD patients with a long duration of disease (more than one year; p < 0.05). The concentrations of 14-3-3 or tau were lowest at the onset or at the end stage of the disease, while the β amyloid concentration remained low throughout the course of the disease. Conclusions: Both 14-3-3 and tau protein are sensitive and specific biomarkers for CJD. The combination of 14-3-3 and β amyloid analysis resulted in the maximum sensitivity, specificity, and positive predictive value. When these biomarkers are used in the diagnosis of CJD, the phase of the disease in which the CSF sample was obtained should be taken into account. Disease duration, dependent on the PrP genotype, also has a significant influence on the level of 14-3-3 and tau in the CSF.

Cerebrospinal fluid biomarkers in Creutzfeldt-Jakob disease.

Creutzfeldt-Jakob disease (CJD) is a rare neurodegenerative disorder. Since the emergence of variant CJD (vCJD) vigilance concerning the diseases incidence has increased and the interest in accurate in vivo diagnosis has augmented. So far, a large number of biomarkers has been investigated as aid in the differential diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) and vCJD. These include, among others, neuron-specific enolase (NSE), microtubuli associated protein Tau, S-100beta, amyloid-beta (Abeta(1-42)) and the 14-3-3 protein. Multiple studies have confirmed that CSF detection of 14-3-3 protein by Western blot was the best single biomarker for sCJD with an average sensitivity and specificity of 92%. Also, in genetic and iatrogenic CJD (iCJD) patients with an average disease duration of less than 1 year, 14-3-3 is the best differential biomarker. Unfortunately, the 14-3-3 protein has a lower sensitivity if the disease duration exceeds beyond 1 year in both sporadic CJD and other CJD types (vCJD, and specific genetic or iatrogenic CJD types).

Affinity Purification of Human τ Proteins and the Construction of a Sensitive Sandwich Enzyme‐Linked Immunosorbent Assay for Human τ Detection

Abstract: Immunoaffinity chromatography with a monoclonal antibody produced against bovine τ protein was used to purify τ proteins from human brain. Fifty grams of brain tissue yielded τ 2 mg of pure τ proteins. The affinity‐purified human τ was used to produce a high‐titered rabbit anti‐human τ serum. The monoclonal anti‐τ antibody and the polyclonal rabbit anti‐τ serum were then used to construct a sandwich enzyme‐linked immunosorbent assay for detection of human τ proteins, with a sensitivity of 1 ng/ml.

Immunoreactivity of presenilin-1 and tau in Alzheimer's disease brain

Mutations in the presenilin-1 gene (PS-1) on chromosome 14 are causative for early-onset familial Alzheimers disease (AD). In order to study the localization of PS-1 in human brain, a polyclonal antibody, SB63, against a N-terminal epitope of PS-1 (25VRSQNDNRERQEHND40), was raised in rabbits and characterized. Immunolabeling with SB63 of formalin-fixed sections of hippocampus from cases of PS-1-linked AD (PS-1 I143T (AD/A), G384A (AD/B)), sporadic AD, and controls showed a predominant neuronal staining pattern with a stronger immunoreactivity in pyramidal neurons. Staining was mainly granular and localized in the neuronal cell body as well as in neuronal processes. In AD some dystrophic neurites surrounding the amyloid plaques were stained, but no immunoreactivity was observed in the amyloid core. Although PS-1 was present in tangle bearing neurons, colocalization of PS-1 and tau could not be detected using immunofluorescence double labeling. Our data indicate that the pattern of PS-1 immunoreactivity in the hippocampus does not substantially differ between PS-1-linked AD, sporadic AD, and controls.

14-3-3 γ-isoform detection distinguishes sporadic Creutzfeldt–Jakob disease from other dementias

We developed a polyclonal antiserum directed to the γ-isoform of the human 14-3-3 protein and compared the immunoreactivity with a commercially available antibody (CG31). We analysed 14-3-3 in 253 cerebrospinal fluid samples blinded for the diagnosis by western blot and ELISA, with a commonly used polyclonal antiserum (Sc-731) and the γ specific antibodies. Our patient population consisted of 52 patients with definite sporadic Creutzfeldt–Jakob disease (sCJD) and 201 patients with a different final diagnosis. We obtained similar sensitivity, ranging from 96% to 98% with all antibodies. Of all the samples that were false positive with Sc-731, 50% were negative with both γ-isoform specific antibodies resulting in a significantly higher specificity (85% v 93%, respectively). If only sCJD and patients with dementia differing from sCJD were analysed we found that 64% of false positives were negative which also resulted in significantly increased specificity and positive predictive value. The γ-isoform specific antibodies strongly improve the specificity of the immunoblot and might improve worldwide acceptance of the use of the 14-3-3 assay in the differential diagnosis of sCJD.

Demonstration of a novel neurofilament associated antigen with the neurofibrillary pathology of Alzheimer and related diseases

A monoclonal antibody, termed NFT200, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer brain. The antigen to which NFT200 is directed was expressed in the paired helical filaments of NFT in sporadic and familial Alzheimer disease (AD), in the straight filaments of NFT in AD, progressive supranuclear palsy and of Pick bodies, and the NFT in several other conditions such as Parkinson-dementia complex of Guam and subacute sclerosing panencephalitis. Granulovacuolar degeneration of AD was also labeled with NFT200. Hirano bodies and amyloid deposits in AD, as well as Lewy bodies of idiopathic Parkinson disease lacked in the antigen. The NFT200-antigen was also expressed as a phosphatase-insensitive antigen in normal neurofilaments found in spinal cord and peripheral nerve axons but was absent from the perikaryal accumulation of neurofilaments induced by aluminum intoxication. Nevertheless, immunoblot studies failed to detect the NFT200 in isolated preparations of the neurofilament proteins, MAP-2, tau, ubiquitin or A4-amyloid peptide. The results indicate that the NFT200 monoclonal antibody is directed against a phosphatase-insensitive epitope of an axonal protein associated with neurofilaments but is labile to isolation and expressed as a stable epitope of a 200 kDa component of NFT.

A monoclonal antibody raised against alzheimer cortex that specifically recognizes a subpopulation of microglial cells

A monoclonal antibody (MAb), termed AMC30, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimers brain. The antigen to which AMC30 is directed was expressed by microglial cells in senile plaques of Alzheimers disease (AD). Microglia in the parenchyma surrounding brain tumors or infarctions, multinuclear giant cells, perivascular and parenchymal macrophages throughout the brain of AIDS patients were also labeled. Different non-nervous system lesions in which macrophages participate were also stained. Microglial cells in normal areas of the cortex or white matter were not labeled with MAb AMC30. The antigen to which AMC30 is directed was not detected in normal bone marrow, lymph nodes, lung, or spleen monocytes or macrophages. The epitope recognized by MAb AMC30 was present after formalin fixation and paraffin embedding. Our findings suggest that this MAb is directed against an antigen that is specifically expressed in a subpopulation of microglial cells and macrophages reactive to various pathological conditions.

Specific monoclonal antibodies against normal microtubule-associated protein-2 (MAP2) epitopes present in Alzheimer pathological structures do not recognize paired helical filaments

SummaryWe have developed monoclonal antibodies that detect normal microtubule-associated protein-2 (MAP2) epitopes in routinely fixed, paraffin-embedded tissue. The somatodendritic distribution of MAP2 in bovine and human nervous tissue was confirmed with several of these antibodies. Furthermore, some of these antibodies immunohistochemically labeled certain pathological structures in Alzheimer brain, especially neurites in senile plaques. Electron microscopic observations, however, indicate that these MAP2 epitopes are not located in the Alzheimer paired helical filaments themselves, but in amorphous granular structures coexistent with them. While the pathological nature of these structures is undetermined, they may represent artefactual modifications of normal cytoskeletal components.

Molecular diagnostic tools in Creutzfeldt-Jakob disease and other prion disorders

Clinical criteria and cerebrospinal fluid biomarkers for the diagnosis of human prion diseases (sporadic, iatrogenic or variant Creutzfeldt–Jakob disease and genetic inherited transmissible spongiform encephalopathies) are now widely available and show a sensitivity and specificity of approximately 98%. Final diagnosis of prion diseases is obtained by post-mortem examination upon identification of the pathological conformer of the prion protein (PrPSc) in the brain. Several diagnostic kits are now available that facilitate the immunochemical measurement of PrPSc. Several new molecular diagnostic techniques, aimed at increasing the sensitivity and specificity of PrPSc detection and at identifying markers of disease other than PrPSc, are the subject of ongoing studies. The aim of these studies is to develop preclinical screening tests for the identification of infected but still healthy individuals. These tests are also essential to investigate the safety of blood or blood-derived products and to ensure meat safety in European countries.