Desenvolvimento de dot-blot para detecção de anticorpos para o vírus da Artrite Encefalite Caprina em caprinos.

Resumo: A técnica de imunodifusão em gel de ágar (IDGA) é empregada mundialmente como método de triagem e monitoramento das fases iniciais de programas de controle das lentiviroses de pequenos ruminantes, mas apesar da boa especificidade, a IDGA pode apresentar resultados falso-negativos. Este trabalho teve como objetivo padronizar o teste Dot-Blot (DB) para a detecção de anticorpos, em caprinos, contra o Lentivírus Caprino (LVC), utilizando antígeno experimental preparado a partir do vírus total, e compará-lo com a IDGA e com ELISA indireto (ELISA-i). Na realização do (DB) a membrana de nitrocelulose (MN) foi disposta num aparelho de blot de 96 poços acrescentando antígeno com uma concentração de 0,5 mg de proteína/poço. Colocaram-se as tiras de MN em tubos de ensaio contendo soro teste diluído (1:50). Após, distribuiu-se o conjugado (peroxidase IgG coelho anti-cabra) diluído 1:500 em PBS-T e revelou-se a MN numa solução de DAB/4-Cloronapthtol. Num total de 327 amostras verificou-se que o ELISA-i detectou 209 caprinos positivos, o DB detectou 200, enquanto a IDGA detectou 144 animais. O DB mostrou concordância de 90,2% (p<0,01) com o Elisa-i. O DB é um teste mais sensível que a IDGA e comparável ao ELISA-i, além de não necessitar da indumentária tecnológica do ELISA-i, podendo ser utilizado em eventos agropecuários ou até mesmo na propriedade. Summary: The imunodifusion in agar gel technique (IDGA) is used worldwide as a screening method of monitoring the initial phases of programs to control lentivirosis of small ruminants. Despite of the good specificity, the IDGA can present false-negative results. The objective of this work was to standardize the Dot-Blot test (DB) for the detention of antibodies against Lentivírus Caprine (LVC), using a prepared experimental antigen from the total virus. The DB was compared with the IDGA and indirect ELISA (ELISA-i) tests. The DB test was accomplished by used a nitrocelulose membrane (MN) in a device of blot of 96 wells and the antigen placed in a concentration of 0.5 mg of protein/well. The strips of MN placed in separated tubes and serum tests added at dilution of 1:50. After that, the conjugated peroxidase IgG rabbit anti-goat diluted 1:500 in PBS-T was incubated for 60 minutes. The DAB/4-Cloronapthtol solution was used to reveal the reaction. From a total of 327 samples, we verified that the ELISA-i detected 209 positives goat, the DB detected 200, while the IDGA 144 animals. The DB showed agreement of 90.2% (p<0.01) with the Elisa-i. The DB is a test more viable than the IDGA and comparable to the ELISA-i test. Besides being more sensible than the IDGA, it does not need the technological equipment of the ELISA-i, being able to be used in animals for events or in the field

Generation and characterization of a recombinant chimeric protein (rCpLi) consisting of B-cell epitopes of a dermonecrotic protein from Loxosceles intermedia spider venom

AbstractA chimeric protein was constructed expressing three epitopes of LiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. This species is responsible for a large number of accidents involving spiders in Brazil. We demonstrated that the chimeric protein (rCpLi) generated is atoxic and that antibodies previously developed in rabbits against synthetic epitopes reactive with rCpLi in ELISA and immunoblot assays. The antibody response in rabbits against the rCpLi was evaluated by ELISA and we have detected an antibody response in all immunized animals. Overlapping peptides covering the amino acid sequence of the rCpLi were synthesized on a cellulose membrane, and their recognition by rabbit anti-rCpLi serum assessed. Three different antigenic regions were identified. The percentage of inhibition of the dermonecrotic, hemorrhagic and edematogenic activities caused by the recombinant protein LiD1r in naïve rabbits was assessed by pre-incubation with anti-rCpLi antibodies. Anti-rCpLi induced good dermonecrotic and hemorrhagic protection. The levels of protection were similar to the antiboides anti-LiD1r. In summary, we have developed a polyepitope recombinant chimeric protein capable of inducing multiple responses of neutralizing antibodies in a rabbit model. This engineered protein may be a promising candidate for therapeutic serum development or vaccination

Micronutrient intake and the probability of nutrient adequacy among children 9-24 months of age : results from the MAL-ED birth cohort study

Peer reviewedPublisher PD

Causal Pathways from Enteropathogens to Environmental Enteropathy: Findings from the MAL-ED Birth Cohort Study

Background Environmental enteropathy (EE), the adverse impact of frequent and numerous enteric infections on the gut resulting in a state of persistent immune activation and altered permeability, has been proposed as a key determinant of growth failure in children in low- and middle-income populations. A theory-driven systems model to critically evaluate pathways through which enteropathogens, gut permeability, and intestinal and systemic inflammation affect child growth was conducted within the framework of the Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) birth cohort study that included children from eight countries. Methods Non-diarrheal stool samples (N = 22,846) from 1253 children from multiple sites were evaluated for a panel of 40 enteropathogens and fecal concentrations of myeloperoxidase, alpha-1-antitrypsin, and neopterin. Among these same children, urinary lactulose:mannitol (L:M) (N = 6363) and plasma alpha-1-acid glycoprotein (AGP) (N = 2797) were also measured. The temporal sampling design was used to create a directed acyclic graph of proposed mechanistic pathways between enteropathogen detection in non-diarrheal stools, biomarkers of intestinal permeability and inflammation, systemic inflammation and change in length- and weight- for age in children 0–2 years of age. Findings Children in these populations had frequent enteric infections and high levels of both intestinal and systemic inflammation. Higher burdens of enteropathogens, especially those categorized as being enteroinvasive or causing mucosal disruption, were associated with elevated biomarker concentrations of gut and systemic inflammation and, via these associations, indirectly associated with both reduced linear and ponderal growth. Evidence for the association with reduced linear growth was stronger for systemic inflammation than for gut inflammation; the opposite was true of reduced ponderal growth. Although Giardia was associated with reduced growth, the association was not mediated by any of the biomarkers evaluated. Interpretation The large quantity of empirical evidence contributing to this analysis supports the conceptual model of EE. The effects of EE on growth faltering in young children were small, but multiple mechanistic pathways underlying the attribution of growth failure to asymptomatic enteric infections had statistical support in the analysis. The strongest evidence for EE was the association between enteropathogens and linear growth mediated through systemic inflammation

Desenvolvimento de dot-blot para detecção de anticorpos para o vírus da Artrite Encefalite Caprina em caprinos.

A técnica de imunodifusão em gel de ágar (IDGA) é empregada mundialmente como método de triagem e monitoramento das fases iniciais de programas de controle das lentiviroses de pequenos ruminantes, mas apesar da boa especificidade, a IDGA pode apresentar resultados falso-negativos. Este trabalho teve como objetivo padronizar o teste Dot-Blot (DB) para a detecção de anticorpos, em caprinos, contra o Lentivírus Caprino (LVC), utilizando antígeno experimental preparado a partir do vírus total, e compará-lo com a IDGA e com ELISA indireto (ELISA-i). Na realização do (DB) a membrana de nitrocelulose (MN) foi disposta num aparelho de blot de 96 poços acrescentando antígeno com uma concentração de 0,5 mg de proteína/poço. Colocaram-se as tiras de MN em tubos de ensaio contendo soro teste diluído (1:50). Após, distribuiu-se o conjugado (peroxidase IgG coelho anti-cabra) diluído 1:500 em PBS-T e revelou-se a MN numa solução de DAB/4-Cloronapthtol. Num total de 327 amostras verificou-se que o ELISA-i detectou 209 caprinos positivos, o DB detectou 200, enquanto a IDGA detectou 144 animais. O DB mostrou concordância de 90,2% (p<0,01) com o Elisa-i. O DB é um teste mais sensível que a IDGA e comparável ao ELISA-i, além de não necessitar da indumentária tecnológica do ELISA-i, podendo ser utilizado em eventos agropecuários ou até mesmo na propriedade. Summary: The imunodifusion in agar gel technique (IDGA) is used worldwide as a screening method of monitoring the initial phases of programs to control lentivirosis of small ruminants. Despite of the good specificity, the IDGA can present false-negative results. The objective of this work was to standardize the Dot-Blot test (DB) for the detention of antibodies against Lentivírus Caprine (LVC), using a prepared experimental antigen from the total virus. The DB was compared with the IDGA and indirect ELISA (ELISA-i) tests. The DB test was accomplished by used a nitrocelulose membrane (MN) in a device of blot of 96 wells and the antigen placed in a concentration of 0.5 mg of protein/well. The strips of MN placed in separated tubes and serum tests added at dilution of 1:50. After that, the conjugated peroxidase IgG rabbit anti-goat diluted 1:500 in PBS-T was incubated for 60 minutes. The DAB/4-Cloronapthtol solution was used to reveal the reaction. From a total of 327 samples, we verified that the ELISA-i detected 209 positives goat, the DB detected 200, while the IDGA 144 animals. The DB showed agreement of 90.2% (p<0.01) with the Elisa-i. The DB is a test more viable than the IDGA and comparable to the ELISA-i test. Besides being more sensible than the IDGA, it does not need the technological equipment of the ELISA-i, being able to be used in animals for events or in the field.200

Imobilização de proteínas do veneno do escorpião Tytius Serrulatus em blenda condutora de Polianilina-Poli(Metacrilato de Hidroxietila) Proteins associated with the venom of the tytius serrulatus scorpion immobilized within polyaniline-poly(Hydroxyethyl Methacrylate) conducting blends

Biossensores têm a função de detectar e analisar tanto quantitativa como qualitativamente a presença de uma certa molécula em um determinado meio, isto com resposta em tempo real e sem etapas intermediárias de processo. Para isso, o biossensor possui sempre em seu conjunto um sistema de reconhecimento biológico que o diferencia de qualquer outro tipo de sensor. O objetivo deste trabalho foi desenvolver hidrogéis condutores a partir da combinação de polianilina com hidrogel poli(metacrilato de hidroxietila) e avaliar alguns aspectos associados à possível utilização deste como base de um biossensor capaz de detectar a presença de frações tóxicas do antígeno do veneno do escorpião Tytius Serrulatus. Hidrogéis condutores foram sintetizados a partir da mistura, em diferentes proporções, de polianilina e poli(metacrilato de hidroxietila) em um solvente comum. A condutividade elétrica (método de quatro pontas), capacidade de inchamento e morfologia (microscopia eletrônica de varredura) foram avaliadas para blendas com diferentes composições. Antígenos relativos ao veneno do escorpião Tytius Serrulatus foram incorporados às blendas condutoras a partir do inchamento dos hidrogéis por soluções aquosas contendo as moléculas de interesse. A imobilização e bioatividade das biomacromoléculas nos hidrogéis foram constatadas através de ensaio imunoabsorvente ELISA cujos resultados foram avaliados por espectroscopia na região do ultravioleta-vísivel. Os resultados mostraram que a incorporação de baixos conteúdos de Pani (20% em massa) em hidrogéis de poli(metacrilato de hidroxietila) foi suficiente para a produção de materiais com elevada condutividade elétrica e grande capacidade de inchamento. Moléculas associadas ao veneno do escorpião Tytius Serrulatus foram imobilizadas com sucesso nos hidrogéis condutores e mantiveram suas capacidades funcionais.<br>Biosensors can detect and analyze quantitatively and qualitatively the presence of a given molecule in a specific environment. Biosensors always have a biological recognition system that distinguishes them from any other kind of sensor. The goals of this work were to develop conductive hydrogels from the combination of polyaniline and poly(hydroxyethyl methacrylate) and to evaluate some important aspects related to the possibility of using these materials as matrices in biosensors that would detect toxic fractions of antigens associated with the venom of the Tytius Serrulatus scorpion. Different conductive blends from poly(hydroxyethyl methacrylate) and polyaniline were produced by dissolving the polymers in a common solvent. The electrical conductivity, morphology and swelling ability were measured the four-probe measuring system, scanning electron microscopy and weight gain in water, respectively. Immobilization and bioactivity of molecules associated with the venom of the Tytius Serrulatus scorpion were evaluated by combining the ELISA immunoassay method and ultraviolet-visible spectroscopy. The results showed that even low concentrations of polyaniline led to high values of electrical conductivity and swelling. The biological tests indicated that the immobilization and bioactivity of the biomacromolecules associated with the venom of the Tytius Serrulatus scorpion were successfully achieved within the conductive hydrogel