Wild rabies virus detection by plaque assay from naturally infected brains in different species

A simple, sensitive and specific plaque assay protocol for the detection of wild type rabies virus in different species is described using confluent monolayers of chicken embryo cells in 6-well plates. Plaques are produced after application of either agarose or Sephadex G-100 overlay onto cell monolayers and incubation for 96 h after virus infection at 37 degreesC. The parameters affecting plaque appearance include cell seeding concentration, overlay composition and time of incubation after infection. Optimal conditions are seeding at a concentration of 4 x 10(6) cell/cm(3), incubation at 37 degreesC in 5% CO2 atmosphere during 96 h, using either 1% agarose or 2% Sephadex G-100 overlays. The described plaque assay would be a new valuable too] in conducting various quantitative investigations, since the chicken embryo cells are susceptible to rabies virus infection from all species studied. (C) 2004 Elsevier B.V. All rights reserved.

Rabies neutralizing antibody detection by indirect immunperoxidase serum neutralization assay performed on chicken embryo related cell line

The aim of this study was to evaluate the indirect immunoperoxidase virus neutralization (IPVN) and mouse neutralization test (MNT) to detect antibodies against rabies virus from vaccinated dogs and cattle. The IPVN was set up for the ability to measure 0.5 International Units/ml (IU) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. IPVN was developed and standardized in chicken embryo related (CER) cell line when 141 dog and 110 cattle sera were applied by serial five-fold dilutions (1:5, 1:25, 1:125) as well as the positive and negative reference controls, all added in four adjacent wells, of 96-well microplates. A 50 µl amount of CVS32 strain dilution containing 50-200 TCID50/ml was mixed to each serum dilution, and after 90 min 50 µl of 3 x 10(5) cells/mlcell suspension added to each well. After five days of incubation, the monolayers were fixed and the IPVN test performed. The correlation coefficient between the MNT and IPVN performed in CER cells was r = 0.9949 for dog sera (n = 100) and r = 0.9307 for cattle sera (n = 99), as well as good specificity (94.7%), sensitivity (87.5%), and agreement (96.6%) were also obtained. IPVN technique can adequately identify vaccinated and unvaccinated animals, even from low-responding vaccinated animals, with the advantage of low cost and faster then MNT standard test.

A novel in situ Polymerase chain reaction hybridisation assay for the direct detection of bovine herpesvirus type 5 in formalin-fixed, paraffin-embedded tissues

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative evaluation of enzyme-linked immunosorbent assay based on crude and purified antigen in the diagnosis of canine visceral leishmaniasis in symptomatic and oligosymptomatic dogs

This study evaluated the performance of crude total antigen (CTA) and fucose-mannose ligand antigen (FML) in an enzyme-linked immunosorbent assay for diagnosis of canine visceral leishmaniasis (CVL). The assays used sera from known negative controls (n = 30), clinically symptomatic (n = 30) and oligosymptomatic (n = 30) parasitologically proven infection (by microscopy). Aspirates of popliteal lymph node from infected canines were colleted to score parasitism and compared with the ELISA results. The study indicated that FML used in ELISA provided high sensitivity for detecting oligosymptomatic dogs (90%) and CTA showed greater sensitivity than FML for symptomatic canines (90%). In oligosymptomatic dogs, specificity was 100% for CTA-ELISA, but in symptomatic dogs, FML specificity was higher (96.7%) than CTA-ELISA (93.3%). A significant correlation was observed between the degree of parasitism and the results obtained in CTA-ELISA. Since no available antigen offers 100% specificity and sensitivity for CVL diagnosis, the choice of antigen used must depend on the aim of the investigation. (C) 2008 Elsevier B.V. All rights reserved.

Detection of the single nucleotide polymorphism (rs2227307) in the human interleukin 8 gene using a pcr-rflp assay

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

PCR associated with agar gel immunodiffusion assay improve caprine arthritis-encephalitis (CAEV) control

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Application of the comet assay in erythrocytes of Oreochromis niloticus (Pisces): a methodological comparison

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evaluation of the antigenotoxicity of polysaccharides and beta-glucans from Agaricus blazei, a model study with the single cell gel electrophoresis/Hep G2 assay

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Genotoxicity assessment of water soluble fractions of biodiesel and its diesel blends using the Salmonella assay and the in vitro MicroFlow (R) kit (Litron) assay

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of chemopreventive activity of glutamine by the comet and the micronucleus assay in mice's peripheral blood

This research has evaluated the effects of enteral supplementation of glutamine in clastogens and genotoxic damages caused by the acute administration of cisplatin. For this. it was utilized Swiss mice distributed in eight experimental groups: control, cisplatin, glutamine, in three different doses and the combination of these with cisplatin. The results show that the glutamine was present in neither genotoxic nor mutagenic activity. When in association with glutamine and cisplatin, in simultaneous treatment, it was verified the frequency decreased of micronuclei and comets. The damage reduction percentages to the micronucleus ranged from 95.4 to 91.8% after 24 h of administration of these compounds and 76.7 to 56.8% after 48 h. In the same time the damage reduction percentages to the comet test ranged from 117.0 to 115.0%. The results suggest that glutamine is capable of preventing genotoxic and mutagenic damage according to the experimental design proposed. (C) 2009 Elsevier B.V. All rights reserved.

Colorimetric enzymatic assay of L-malic acid using dehydrogenase from baker's yeast

A colorimetric method has been developed and optimized to measure L-malic acid in samples of fruit juices and wine. This method is based on oxidation of the analyte, catalyzed by malate dehydrogenase (MDH) from dry baker's yeast, and in combination with the reduction of a tetrazolium salt (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). In the present study, the method exhibited sensitivity in the range of 500-4000 mu M of L-malic acid in the reaction cuvette, with the lower detection limit of 6.7-10(-2) g/L, the upper limit of 53.6.10(-2) g/L and a maximum standard deviation of only 2.5 % for the analyzed samples. The MDH activity from baker's yeast was also optimized, the enzyme showed a high stability at pH=8.0-9.0 and the activity was maintained completely at temperatures up to 40 degrees C for 1 hour. The results show that the colorimetric method using enzymatic preparations from dry baker's yeast is a simple and low-cost method with possibility of wide application.

Development of a rapid one-step immunochromatographic assay for HCV core antigen detection

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Differential Toxicity of Disperse Red 1 and Disperse Red 13 in the Ames Test, HepG2 Cytotoxicity Assay, and Daphnia Acute Toxicity Test

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)