Evaluation of Fluorescence Microsphere Immunoassay for Detection of Antibodies to Rift Valley Fever Virus Nucleocapsid Protein and Glycoproteins.

Rift Valley fever virus (RVFV) is a mosquito-borne, zoonotic virus that infects ruminants, including cattle, sheep, goats, camels, and buffalo. Multiplexing diagnostic assays that can simultaneously detect antibodies against multiple RVFV antigens offer a high-throughput test for disease surveillance and vaccine evaluations. We describe the improvement and evaluation of a previously developed fluorescence microsphere immunoassay (FMIA) for the detection of IgG and IgM antibodies against the RVFV glycoprotein (Gn) and the immunogenic nucleocapsid protein (Np). Well-characterized vaccinated and experimentally infected ruminant sera were used for the evaluation of the assay. Recombinant viral proteins were produced and then coupled to polystyrene magnetic beads for analysis using the Luminex MAGPIX system with xMAP technology. The FMIA was performed in parallel with virus neutralization tests. Our results revealed the highest median fluorescence intensity (MFI) values for the detection of IgG antibodies against RVFV Np, indicating that this antigen would be a good candidate for a screening assay. The Np and Gn targets could differentiate infected animals from animals vaccinated with a candidate subunit vaccine formulation based on the RVFV Gn and Gc proteins. The results presented in this report demonstrate that FMIA provides a rapid and robust serological diagnostic tool for the detection of antibodies against RVFV. The targets developed in this assay provide the basis for the development of a companion diagnostic test for an RVFV Gn/Gc subunit vaccine that is capable of differentiating infected from vaccinated animals (DIVA), as well as a multiplex serodiagnostic assay that can simultaneously screen for several ruminant diseases.

The vaccines consistency approach project: an EPAA initiative.

The consistency approach for release testing of established vaccines promotes the use of in vitro, analytical, non-animal based systems allowing the monitoring of quality parameters during the whole production process. By using highly sensitive non-animal methods, the consistency approach has the potential to improve the quality of testing and to foster the 3Rs (replacement, refinement and reduction of animal use) for quality control of established vaccines. This concept offers an alternative to the current quality control strategy which often requires large numbers of laboratory animals. In order to facilitate the introduction of the consistency approach for established human and veterinary vaccine quality control, the European Partnership for Alternatives to Animal Testing (EPAA) initiated a project, the "Vaccines Consistency Approach Project", aiming at developing and validating the consistency approach with stakeholders from academia, regulators, OMCLs, EDQM, European Commission and industry. This report summarises progress since the project's inception.

3-(1-piperazinyl)-4,5-dihydro-1H-benzo[g]indazoles: high affinity ligands for the human dopamine D4 receptor with improved selectivity over ion channels.

3-(4-Piperidinyl)-5-arylpyrazoles, such as 1, were selective for the cloned human dopamine D4 receptor (hD4), but also showed affinity at voltage sensitive calcium, sodium and potassium ion channels. A combination of substituent changes to reduce the basicity of the piperidine nitrogen and conformational restriction to give 4,5-dihydro-1H-benzo[g]indazoles reduced this ion channel affinity at the expense of selectivity for hD4 over other dopamine receptors. Incorporation of piperazine into the 4,5-dihydro-1H-benzo[g]indazoles in place of piperidine gave a novel series of high affinity, selective, orally bioavailable hD4 ligands, such as 16, with improved selectivity over ion channels.

Fatal combined defects in mitochondrial multienzyme complexes in two siblings.

A female child suffering from intrauterine growth retardation was born by caesarean section at 32 weeks. In the immediate newborn period there was a metabolic acidosis but this resolved. Hypotonia, muscular weakness and poor respiratory effort were evident and the child died at 6 days of age. A previous male sibling had died at 3 months of age after similar symptoms with seizures and a dysmyelination disorder. Post-mortem examination of both children showed damage to the basal ganglia. Defects in the activities of the pyruvate dehydrogenase complex, cytochrome oxidase and succinate cytochrome c reductase were found in cultured skin fibroblasts. Similar defects were found in isolated muscle mitochondria but not in isolated liver mitochondria from the patient. Immunoblotting for cytochrome oxidase showed that the multienzyme complex was not assembled in muscle and skin fibroblast mitochondria, but was assembled in liver mitochondria. Similar results were obtained in cultured skin fibroblast mitochondria for complex I of the mitochondrial respiratory chain. This is the first occasion that multiple defects have been demonstrated both in tissue and in culture skin fibroblasts in mitochondrial respiratory chain complexes.

Determination of the cDNA sequence for the human mitochondrial 75-kDa Fe-S protein of NADH-coenzyme Q reductase.

A human-hepatoma cDNA lambda gt11 expression library was probed with an antibody to holoenzyme complex I (NADH-CoQ reductase) of the respiratory chain. One of the 30 antibody positive clones was purified to homogeneity, amplified by the polymerase chain reaction (PCR), subcloned and sequenced. It proved to be highly similar to the cDNA sequence for the bovine 75-kDa Fe--S protein. Using the sequence obtained from this library, both sense and antisense oligonucleotides were constructed and used to probe a human kidney cDNA library using PCR amplification with oligonucleotides that flank the polylinker region of the lambda phage. Two further cDNA clones were obtained which overlapped and covered the entire cDNA sequence of 2526 bp. The encoded protein of 727 amino acids has 21 amino acids that differ from the bovine-protein sequence. Northern blot analysis of mRNA from fibroblasts of complex-I deficient patients revealed no abnormalities. We show that this Fe--S protein has significant similarity with (a) the gamma chain of the hydrogen hydrogenase of Alcaligenes eutrophus and (b) the A chain of the formate dehydrogenase of Methanobacterium formicum.

Mitochondrial autoantigens in primary biliary cirrhosis. Association of disease-specific determinants with a subunit of complex I (NADH-ubiquinone reductase) of the inner mitochondrial membrane.

Anti-mitochondrial autoantibodies (AMA) from patients with primary biliary cirrhosis (PBC) were analysed for fine specificity by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Inhibition ELISA showed that complex I (NADH-ubiquinone reductase) from beef heart mitochondria completely inhibited the binding of AMA to mitochondrial inner membranes (SMP), indicating that the major mitochondrial antigens are located in complex I. Immunoblot analysis of beef heart SMP, complex I and the iron sulphur (IP) subfraction of complex I revealed several antigens, one of which (75 kDa) reacted with all PBC sera but not with the additional autoimmune sera tested. Resolution of SMP or complex I by two-dimensional electrophoresis yielded in both preparations a polypeptide of 75 kDa with an isoelectric point of 6.4, which reacted with PBC serum and with rabbit antisera raised against the 75,000 subunit of complex I. In immunoblot experiments, the antigenicity of the 75,000 polypeptide in SMP, complex I, and the IP subfraction is increased by prior reduction of the sample with mercaptoethanol. This suggests a similarity to the PBC-specific 'M-2' antigen, which is also sensitive to sulphur reagents. The data indicate that the 75 kDa polypeptide of complex I is a major mitochondrial antigen binding AMA in PBC sera, and allows us to identify the location and probable function of the PBC antigen.

Evidence that the major primary biliary cirrhosis-specific mitochondrial autoantigen is a subunit of complex I of the respiratory chain.

Primary biliary cirrhosis (PBC)-specific antigens were purified from beef heart mitochondria by immunoaffinity chromatography. Three major polypeptides (75, 60, and 40 kDa) were detected in the purified antigen fraction both by Coomassie blue staining and by western blot analysis. The 75 kDa antigen was identified as a subunit of Complex I (NADH-ubiquinone reductase) by the following criteria: (1) antibodies against the purified 75 kDa subunit of beef heart Complex I react with the immunoaffinity-purified 75 kDa antigen. (2) the 75 kDa subunit present in isolated Complex I, like that in the immunoaffinity-purified antigen, reacts with PBC sera only after reduction with mercaptoethanol, and (3) the 75 kDa antigen is enriched in isolated Complex I. A relationship between the 75 kDa and the 60 and 40 kDa antigens is suggested, since optimal binding of anti-mitochondrial autoantibodies (AMA) to the latter antigens also requires prior reduction with mercaptoethanol. A fourth major antigen (70 kDa) was also detected by western blot analysis, but only in samples that had not been boiled prior to electrophoresis. This antigen, which is also present in isolated Complex I, resembles the 75, 60, and 40 kDa antigens in its response to mercaptoethanol and its reaction with antibodies against the 75 kDa subunit of Complex I. A scheme is presented which relates all of the PBC antigens to the parent 75 kDa subunit of Complex I, probably as proteolytic products of the latter.

Photolabelling of a mitochondrially encoded subunit of NADH dehydrogenase with [3H]dihydrorotenone.

Mitochondrial NADH dehydrogenase from bovine heart was photolabelled with the inhibitor [3H]dihydrorotenone. A constituent of the hydrophobic domain of the enzyme of Mr 33,000 was the major site of labelling. The identity of this protein with the mitochondrially encoded ND-1 gene product was established by immunoblotting and immunoprecipitation with an antiserum raised to the expected C-terminal sequence of the human ND-1 gene product.