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The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15™ cells using the expression vector pQE-30™. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.

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We have developed an indirect enzyme-linked immunosorbent assay for detection of anti-dengue virus (DENV) immunoglobulin G antibodies using four recombinant DENV envelope polypeptides as antigens, which demonstrated a sensitivity of 89.4% and a specificity of 93.3%. These easily produced antigens are a feasible, cost-effective alternative for generating reagents for dengue serological tests.The accurate and efficient diagnosis of dengue is important for clinical care, surveillance, pathogenesis studies, and vaccine research. Dengue viruses consist of four serotypes (DENV1 to -4) (17) and cause a spectrum of disease ranging from self-limited illness (dengue fever) to more severe forms of disease (dengue hemorrhagic fever/dengue shock syndrome) (25). Dengue is one of the most important mosquito-borne viral diseases in the world (10), with 2.5 to 3 billion people at risk for infection and tens of millions of dengue cases annually. Nonetheless, the disease remains significantly underreported (15). Although numerous commercial kits for serological diagnosis of dengue are available, some of which have shown good sensitivity and specificity (3,8), their cost poses a financial burden to many countries where dengue is endemic.Enzyme-linked immunosorbent assay (ELISA)-formatted tests to detect anti-DENV immunoglobulin G (IgG) antibodies have been developed as an alternative method to the hemagglutination inhibition test, which has served as the "gold standard" for the diagnosis and classification of DENV infections (4,5,12,18,25). Moreover, an IgG-ELISA was reported to be an accurate and reliable assay for characterization of human immune responses in primary and secondary DENV infections (18). However, these ELISAs require viral antigen produced, in limited quantities, by growing DENV in cell culture or in suckling mouse brain, which is insufficient for the large-scale serological screening made necessary by the spread of the disease. Furthermore, the use of whole viruses or crude extracts represents potential health hazards through exposure to infectious virus particles. To overcome this problem, we developed and evaluated an in-house ELISA for detecting anti-DENV IgG antibodies using as the antigen a mixture of DENV1 to -4 recombinant polypeptides, as its use for IgM capture has been previously demonstrated (23). Fragments of approximately 500 bp from the 5Ј end (nucleotides 1093 through 1585) of the DENV1 to -4 E genes were amplified by reverse transcription-PCR (RT-PCR) and cloned into an expression vector, and the immunogenic E polypeptides (ϳ25 kDa) were easily expressed in Escherichia coli (7, 23).The 271 sera used in this study were from the Flavivirus Laboratory, Oswaldo Cruz Institute/FIOCRUZ, Brazil (1998Brazil ( to 2005. Laboratory-positive DENV infection was defined in patients experiencing a febrile illness consistent with dengue according to WHO criteria (25), in accordance with which infection was confirmed by DENV isolation (9), detection of DENV RNA by RT-PCR (16), detection of anti-DENV IgM antibodie...

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Objetivo: Este trabalho identifica e avalia os riscos do processo produtivo de obtenção da partícula calibradora (PC) e controle positivo (CP), dois importantes insumos do KIT NAT HIV/HCV de Bio-Manguinhos. Metodologia: Os riscos identificados referem-se ao processo produtivo visando à qualidade dos insumos (PC e CP). Foram utilizadas de forma complementar duas ferramentas de análise de risco: PHA -Preliminary Hazard Analisys (Análise Preliminar de Perigos) e HACCP-Hazard Analysis and Critical Control Points (Análise de Perigos e Pontos Críticos de Controle). A PHA foi utilizada em um primeiro momento para, a partir do fluxograma do processo produtivo, identificar eventos que possam influenciar negativamente a qualidade dos produtos. Estes eventos foram classificados em uma matriz de risco. Delimitamos na matriz de risco uma região associada aos riscos mais importantes. As etapas do processo produtivo onde encontramos os riscos mais importantes foram consideradas Pontos Críticos do Processo (PCC). Os PCC foram estudados mais detalhadamente através da segunda ferramenta, o HACCP. A utilização desta ferramenta permite estabelecer os limites críticos, um sistema de monitoramento e medidas de controle para todos os PCC. Desta forma, a qualidade destes produtos pode ser avaliada em tempo real, ou seja, durante o processo produtivo e antes da análise pelo controle de qualidade. Resultado: Foi proposto e validado um fluxograma para o processo produtivo baseado em duas plataformasa) plataforma procariótica (na qual são obtidos os plasmídeos que contem a informação genética dos produtos, concentrados e com alto grau de pureza através da transformação e cultivo de bactérias competentes) e b) plataforma eucariótica