Production and characterisation of a candidate hyper-immune serum for the replacement of the Bordetella pertussis mouse antiserum Biological Reference Preparation.

For acellular pertussis (aP) vaccines, the current European Pharmacopoeia (Ph. Eur.) monograph Pertussis vaccine (acellular, component, adsorbed) (1356) requires an immunogenicity assay in mice or guinea pigs to assess the potency of each lot of vaccine (Ph. Eur. general method 2.7.16. Assay of pertussis vaccine (acellular)). This biological assay, carried out on the final bulk of the vaccine lot, is based on the measurement of the specific antibody response to the 5 antigenic components (pertussis toxin (PT), Fimbrial haemagglutinin (FHA), pertactin (PRN) and Fimbriae 2 and 3 (FIM2/3)) that are present in the combined aP vaccines. In the mouse assay, serum antibody levels are measured by ELISA. The immunogenicity of a vaccine under test is estimated versus a homologous reference vaccine and a reference antiserum e.g. the first Ph. Eur. Biological Reference Preparation for Bordetella (B.) pertussis mouse anti-serum (BRP1), established in 1998, is used to normalise the titre of antibodies (expressed in ELISA Units (ELU)/mL). In anticipation of the depletion of BRP1 stocks, a project was launched in 2013 by the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM) in order to establish a new standardised reference serum. The project, referred to herein as BSP129, was conducted in 2 phases: 1) the production and characterisation of a mouse serum pool (using a multicomponent aP vaccine marketed in Canada similar to the vaccine used in the BRP1 production as immunogen) and of candidate BRP batches (cBRPs) and 2) an international collaborative study aimed at calibrating the cBRPs in terms of antibody levels against PT, FHA, PRN and FIM2/3. This article presents the design and results of the first phase of the collaborative study to establish the optimal conditions for immunisation and bleeding of mice in order to produce a large pool of hyper-immune serum against the 5 antigens. After the characterisation of this pool, cBRP pilot lots were manufactured by freeze-drying diluted solutions of the hyper-immune serum pool. The pilot lots were then characterised in two Official Medicines Control Laboratories (OMCLs) for their antibody contents against aP vaccine antigens using in-house ELISA (based on methods developed by 2 European vaccine manufacturers) and Multiplex Immunoassay (MIA) methods. The antibody titres recovered demonstrated that a dilution factor of 1/40 could be considered for the scaled-up manufacture of candidate reference preparations (cBRPs). Three batches (15 000 vials) of cBRP were manufactured and fully characterised. In light of the data obtained, and although titration results between the ELISA methods were sometimes discrepant, it was agreed that the establishment study (phase 2) could be launched. Real-time and accelerated stability studies were also included in the first study phase to document the stability of the cBRPs in freeze-dried form and after reconstitution and storage at -20°C±5°C. The results showed that the stability of the freeze-dried cBRPs at usual storage and shipment temperatures is acceptable and that reconstituted cBRP solutions are stable for 12 months at -20°C±5°C. It could therefore be recommended to freeze small aliquots of the 1 mL solution obtained by the reconstitution of one BRP vial in order to store them for use in separate assays. With the application of this strategy, the stocks of the BRP1 replacement batches should cover the needs of OMCLs and manufacturers for at least the next decade.

Risk assessment of dimethylfumarate residues in dwellings following contamination by treated furniture.

Recently, numerous cases of dermatitis induced by dimethylfumarate (DMFu) have been reported in Europe. DMFu has been used to prevent mold development in various items, although it is not registered as a biocide. In France, from October 2008 to December 2009, more than 100 cases were reported. Despite a ban on articles containing DMFu and the removal of potentially contaminated products, some people were still suffering from dermatitis or other health problems. The French Agency for Food, Environmental and Occupational Health & Safety was mandated to assess whether the existence in the past of DMFu-contaminated items in dwellings could continue to pose a threat to the health of inhabitants. A risk assessment was performed based on the classical risk analysis approach for environmental contaminants. Hazard assessment of DMFu with regard to its sensitizing properties was performed, based on human case reports collected in France between January 2009 and February 2010. For around half of the 132 individual cases reported, the causal link to DMFu was considered at least probable. An Organisation for Economic Co-Operation and Development (OECD) local lymph node assay performed in a study on mice showed strong sensitizing potential for DMFu. Exposure was assessed by measuring DMFu in items sampled in preselected dwellings. These investigations demonstrated that DMFu exposure can persist after removal of the primary contaminated items. We therefore concluded that there was clearly a risk of skin reactions in patients previously sensitized to DMFu. Furthermore, the available data do not support the existence of significant health effects through the respiratory route.

Differential phosphorylation of tau proteins during kitten brain development and Alzheimer's disease.

Differential distribution and phosphorylation of tau proteins were studied in developing kitten brain by using several antibodies, and was compared to phosphorylation in Alzheimer's disease. Several antibodies demonstrated the presence of phosphorylated tau proteins during kitten brain development and identified pathological structures in human brain tissue. Antibody AD2, recognized tau in kittens and adult cats, but reacted in Alzheimer's tissue only with a pathological tau form. Antibody AT8 was prominent in developing kitten neurons and was found in axons and dendrites. After the first postnatal month this phosphorylation type disappeared from axons. Furthermore, dephosphorylation of kitten tau with alkaline phosphatase abolished immunoreactivity of AT8, but not that of AD2, pointing to a protection of the AD2 epitope in cats. Tau proteins during early cat brain development are phosphorylated at several sites that are also phosphorylated in paired helical filaments during Alzheimer's disease. In either event, phosphorylation of tau may play a crucial role to modulate microtubule dynamics, contributing to increased microtubule instability and promoting growth of processes during neuronal development or changing dynamic properties of the cytoskeleton and contributing to the formation of pathological structures in neurodegenerative diseases.

Binding specificity of monoclonal antibody AD2: influence of the phosphorylation state of tau.

Using recombinant human tau protein phosphorylated by a brain extract and the glycogen synthase kinase-3beta in the absence or the presence of heparin, we showed that phosphorylation-dependent antibody AD2 recognition only requires phosphorylated Ser-396. By the Spot multiple peptide synthesis method, we showed that Tyr-394, Ser(P)-396 and Pro-397 are critical for AD2 binding. A decrease in the binding of AD2 was observed with increasing phosphorylation of residues in the vicinity of Ser(P)-396.

ELISA-quantitation of phosphorylated tau protein in the Alzheimer's disease brain.

A reliable, sensitive and specific sandwich ELISA for the quantitation of paired helical filament (PHF) tau in human brain was developed using well-defined monoclonal antibodies. We examined rapid-autopsy-derived brain tissue from 21 neuropathologically confirmed Alzheimer's disease (AD) patients and 14 nondemented controls, matched for age, sex and postmortem delay times. We demonstrated significant elevations of phosphorylated tau levels in the frontal and parietal cortex as well as in the hippocampus of AD patients as compared to the nondemented controls. No difference was observed in the cerebellum. Phosphorylated tau levels measured by ELISA were significantly correlated with the presence or absence of neurofibrillary tangles.

Tau and apo E in CSF: potential aid for discriminating Alzheimer's disease from other dementias.

To evaluate the usefulness of tau proteins as biological markers in the diagnosis of dementia of the Alzheimer type (DAT), we analyzed the concentration of tau proteins in 253 cerebrospinal fluid (CSF) samples from patients with or without neurological disorders. Our study showed a significant increase of the mean CSF tau concentration in DAT patients compared with that from non-DAT patients. Interestingly, a significative decrease of CSF tau in patients with frontotemporal dementia was found. We also observed a positive correlation between the CSF-tau concentration and the number of apoepsilon4 alleles. The CSF apolipoprotein E concentration was evaluated and revealed no variation between the groups, although we observed a significant correlation between CSF tau and apolipoprotein E in DAT patients.

AD2, a phosphorylation-dependent monoclonal antibody directed against tau proteins found in Alzheimer's disease.

Alzheimer's disease is characterized by an intraneuronal aggregation of hyperphosphorylated tau proteins into paired helical filaments. The hyperphosphorylation of tau proteins induces a decrease in their electrophoretic mobility, resulting in a pathological tau triplet referred to as tau 55, 64 and 69 or tau-PHF. We have developed monoclonal antibodies directed against this pathological tau triplet. In the present article, we report the properties of antibody AD2, which detects the hyperphosphorylated tau proteins forming paired helical filaments during Alzheimer's disease. Using immunoblotting, AD2 exclusively labeled the tau triplet, while normal tau proteins from control cases were not immunodetected. Furthermore, AD2 is highly specific in that it was able to detect the triplet not only in tau preparations but also in total brain homogenates from Alzheimer's disease patients. The binding of this monoclonal antibody to tau proteins is phosphorylation dependent. Characterization of this antibody allowed us to identify its epitope as containing phosphorylated Ser-396 with the participation of phosphorylated Ser-404. AD2 was also shown to label normal tau proteins from rapidly processed brain tissues, but its epitope is rapidly dephosphorylated during postmortem intervals. However, in autopsic brains, AD2 still represents a valuable tool to investigate neurofibrillary degeneration at the biochemical and immunocytochemical levels.

Phorbol ester enhances phosphorylated tau protein immunoreactivity in neuronal cultures.

One of the hallmarks of Alzheimer's disease (AD) is neurofibrillary degeneration which results from the aggregation of phosphorylated tau proteins into paired helical filament (PHF) structures. AD2 is a new monoclonal antibody raised against PHF tau which detects neurofibrillary tangles in AD brain. In primary neuronal cultures, phorbol ester treatment induced a time- and dose-dependent increase in AD2 immunoreactivity quantified by laser confocal microscopy and immunoblottings. Alkaline phosphatase treatment reversed these immunocytochemical changes. These results suggest that the modifications of neuronal metabolism induced by phorbol ester including protein kinase C activation produce an increase in phosphorylated tau immunoreactivity.

New immunoassay for the mapping of neurofibrillary degeneration in Alzheimer's disease using two monoclonal antibodies against human paired helical filament tau proteins.

Monoclonal antibodies against human paired helical filament tau (PHF-tau) proteins were produced. Two of these antibodies, AD1 and AD2, were shown by immunoblot to be directed against distinct hyperphosphorylated epitopes of the PHF-tau proteins. Using AD1 and AD2, an antigen-capture ELISA specific for PHF-tau proteins was developed and used to map the neurofibrillary degeneration of several Broadmann areas from an Alzheimer's disease patient. The results confirm that the neurofibrillary degeneration predominates in parietal and temporal lobes.

Biochemical characterization of Tau proteins during cerebral aging of the lemurian primate Microcebus murinus.

Tau proteins extracted from the brain of 12 adult microcebes ranging from 2 to 9 years old were characterized by Western blots, using immunological probes against normal and pathological human Tau proteins. In microcebes, the molecular weight of Tau proteins increases during aging, with variants of 52-54, 64, 67 kDa in the young adult and variants of 60 and 70 kDa in the oldest animal studied. The increase of the apparent molecular weight is due to a change of conformation and a stabilization in the "hyperphosphorylated" state, as revealed with phosphorylation-dependent monoclonal antibodies Tau-1 and AD2. Furthermore, AD1 specifically detected Alzheimer-type epitopes on the 60 kDa Tau isoform from a very old microcebe. These results suggest that Microcebus murinus is an interesting model for the study of the biochemical dysfunctions that occur in the human brain during aging and Alzheimer disease.

Immunogold labelling of paired helical filaments and amyloid fibrils by specific monoclonal and polyclonal antibodies.

Senile plaque and paired helical filament (PHF) formation are characteristic of Alzheimer's disease, but the mechanisms leading to these lesions still remain unclear. To understand them better, we have performed different immunolabellings of amyloid protein and PHF. We describe a very specific immunodetection of PHF with AD2, a monoclonal antibody directed against a hyperphosphorylated epitope of PHF-tau, and use double immunolabelling to show that PHF and plaque amyloid are discretely labelled by different antibodies. We also discuss different mechanisms of PHF maturation.