Neste trabalho foram comparados os valores de absorbância dos extratos, severidade dos sintomas e biomassa das plantas de abobrinha-de-moita (Cucurbita pepo L) cv. Caserta, de abóbora (C. moschata Duch.) cv. Menina Brasileira e de melancia (Citrullus lanatus (Thunb.) Matsum. & Nakai) cv. Crimson Sweet infectadas com três estirpes fracas (PRSV-W-1 J PRSV-W-2 e PRSV-W-3) e uma estirpe severa (PRSV-W-C) do Papaya ringspot virus - type W (PRSV-W). As plantas-teste foram inoculadas em estádio cotiledonar e mantidas em condições de casa de vegetação. Aos 7, 14, 21, 28 e 35 dias após a inoculação (DAI), amostras de discos foliares foram coletadas das plantas, maceradas em tampão PBS, na proporção de 1:20 (p:v) e armazenadas a -20°C. Testes preliminares comparando as técnicas PTA-ELlSA ("Plate Trapped Antigen"-"Enzyme Linked Immunosorbent Assay") e DAS-ELISA ("Double Antibody Sandwich"- ELISA), realizados com diferentes concentrações do PRSV-W purificado, entre 32 e 4096 ng mL-1, mostraram alta correlação entre os valores de absorbância a 405 nm obtidos e as concentrações do vírus para os dois testes. Diante desse resultado, o PTA- ELISA foi adotado nos experimentos de estimativa da concentração viral. A severidade dos sintomas foi avaliada por uma escala dê notas de 1 (sem mosaico) a 5 (mosaico com bolhas, deformações foliares intensas e atrofiamento da planta). A biomassa da parte aérea das plantas foi avaliada através da pesagem das massas fresca e seca aos 40 DAI. Os resultados obtidos mostraram que as concentrações das estirpes fracas, baseadas nos valores de absorbância do PTA-ELlSA, foram menores do que a observada com a estirpe severa nas três espécies estudadas. Houve também uma variação nos picos de concentração virais das estirpes fracas e severa entre os dois experimentos realizados. As estirpes fracas não causaram sintomas de mosaico nas plantas-teste das três espécies estudadas (nota 1). A estirpe PRSV-W-C causou sintomas extremamente severos nas plantas de abobrinha-de-moita e melancia (nota 5) e sintomas relativamente fracos de mosaico nas plantas de abóbora 'Menina Brasileira' (nota 2). Os valores de biomassa das plantas de abobrinha-de-moita e melancia infectadas pelas estirpes fracas sofreram reduções que variaram de 1,7 % a 12,4 %, quando comparados aos das plantas sadias. Já os valores de biomassa das plantas infectadas pela estirpe severa sofreram reduções mais drásticas, variando de 29 % a 74 %. Os valores de biomassa das plantas de abóbora 'Menina Brasileira' infectadas com as estirpes fracas e severa foram ligeiramente superiores aos das plantas sadias. / not available

Rotavírus têm sido identificados mundialmente como o mais importante agente etiológico de diarréias agudas não-bacterianas em animais jovens de várias espécies, incluindo a humana. Foi desenvolvido e avaliado um método de ELISA tipo “duplo-sanduíche" para a detecção de rotavírus a partir de material fecal. Para tanto, a amostra NCDV de rotavírus do grupo A foi propagada em cultivo celular com células MA-104. O vírus foi concentrado por ultracentrifugação e inoculado em coelhos e carneiros. Em seguida, as frações IgG, oriundas de amostras de soro dos animais, foram purificadas por cromatografia de troca iônica e absorvidas com soro total de ambas espécies animais, utilizando-se polímero de glutaraldeído, de modo a eliminar reações inespecíficas. A presença do rotavírus foi detectada pela IgG de carneiros e revelada pela IgG de coelho, usando como conjugado IgG de cabra anti-IgG de coelho conjugada à peroxidase. Os valores de diluição dos componentes do ELISA e o valor do ponto-de-corte foram definidos usando-se 26 amostras fecais (13 positivas e 13 negativas) de leitões, tendo como prova padrão a eletroforese em gel de poliacrilamida (PAGE). Aplicado a um painel constituído de 86 amostras fecais diarréicas de leitões, os resultados do ELISA foram: 100% de sensibilidade; 98,79% de especificidade, com uma concordância de 98,83%. A variância entre 86 repetições da mesma amostra foram 0,001 (para a amostra positiva) e 0,0002 (para a amostra negativa). Estes resultados demonstram que este ELISA é um teste sensível e específico para o diagnóstico de rotavírus a partir de material fecal. / Rotaviruses have been identified worldwide as a major etiologic agent of acute nonbacterial diarrhea in the young of many species, including humans. In this investigation was developed and evaluated a “double-sandwich" antibody ELISA method for detection of rotavirus from stool specimens. For that, the NCDV strain of rotavirus group A was serially cultivated in MA-104 cell culture. The virus was concentrated by ultra-centrifugation and inoculated in rabbits and sheeps. After that, the IgG of serum samples of the animals was purified by ion-exchange chromatography and absorbed with whole serum of both animal species using a glutaraldehyde polymer, in order to eliminate inespecific reactions. The presence of rotavirus was detected by the sheep’s IgG and revelated by the rabbit’s IgG, using a anti-rabbit IgG peroxidase conjugate developed in goat. The values of diluition of the components of the ELISA and the cut-off value were defined using 26 fecal samples (13 positive and 13 negative) of piglets. Following this procedure, the test was employed in a panel of 86 fecal samples from piglets with diarrhea, using as standard the polyacrilamide gel electrophoresis (PAGE) test. The results of the ELISA were: 100% of sensivity; 98.79% of specificity, with an agreement of 98.83%. The variance between 86 repetitions of the same sample were 0.001 (for one positive sample) and 0.0002 (for one negative sample). These results showed that this ELISA is an sensitive and specific screening test for rotavirus diagnosis from fecal material.

Neste trabalho foram comparados os valores de absorbância dos extratos, severidade dos sintomas e biomassa das plantas de abobrinha-de-moita (Cucurbita pepo L) cv. Caserta, de abóbora (C. moschata Duch.) cv. Menina Brasileira e de melancia (Citrullus lanatus (Thunb.) Matsum. & Nakai) cv. Crimson Sweet infectadas com três estirpes fracas (PRSV-W-1 J PRSV-W-2 e PRSV-W-3) e uma estirpe severa (PRSV-W-C) do Papaya ringspot virus - type W (PRSV-W). As plantas-teste foram inoculadas em estádio cotiledonar e mantidas em condições de casa de vegetação. Aos 7, 14, 21, 28 e 35 dias após a inoculação (DAI), amostras de discos foliares foram coletadas das plantas, maceradas em tampão PBS, na proporção de 1:20 (p:v) e armazenadas a -20°C. Testes preliminares comparando as técnicas PTA-ELlSA ("Plate Trapped Antigen"-"Enzyme Linked Immunosorbent Assay") e DAS-ELISA ("Double Antibody Sandwich"- ELISA), realizados com diferentes concentrações do PRSV-W purificado, entre 32 e 4096 ng mL-1, mostraram alta correlação entre os valores de absorbância a 405 nm obtidos e as concentrações do vírus para os dois testes. Diante desse resultado, o PTA- ELISA foi adotado nos experimentos de estimativa da concentração viral. A severidade dos sintomas foi avaliada por uma escala dê notas de 1 (sem mosaico) a 5 (mosaico com bolhas, deformações foliares intensas e atrofiamento da planta). A biomassa da parte aérea das plantas foi avaliada através da pesagem das massas fresca e seca aos 40 DAI. Os resultados obtidos mostraram que as concentrações das estirpes fracas, baseadas nos valores de absorbância do PTA-ELlSA, foram menores do que a observada com a estirpe severa nas três espécies estudadas. Houve também uma variação nos picos de concentração virais das estirpes fracas e severa entre os dois experimentos realizados. As estirpes fracas não causaram sintomas de mosaico nas plantas-teste das três espécies estudadas (nota 1). A estirpe PRSV-W-C causou sintomas extremamente severos nas plantas de abobrinha-de-moita e melancia (nota 5) e sintomas relativamente fracos de mosaico nas plantas de abóbora 'Menina Brasileira' (nota 2). Os valores de biomassa das plantas de abobrinha-de-moita e melancia infectadas pelas estirpes fracas sofreram reduções que variaram de 1,7 % a 12,4 %, quando comparados aos das plantas sadias. Já os valores de biomassa das plantas infectadas pela estirpe severa sofreram reduções mais drásticas, variando de 29 % a 74 %. Os valores de biomassa das plantas de abóbora 'Menina Brasileira' infectadas com as estirpes fracas e severa foram ligeiramente superiores aos das plantas sadias. / not available

A paracoccidioidomicose (PCM) é caracterizada como uma micose profunda e que acomete, principalmente, indivíduos do sexo masculino e de regiões agrícolas. A infecção ocorre através da inalação de propágulos fúngicos presentes no ambiente, atingindo inicialmente os pulmões e podendo se disseminar para outros órgãos. O diagnóstico precoce da doença previne a formação de sequelas e garante que o trabalhador dê continuidade a sua atividade de vida diária. Os testes de intradermorreação, imunodifusão dupla e ELISA são os mais utilizados no diagnóstico da PCM, no entanto, podem apresentar reatividade cruzada com outras doenças, como Histoplasmose, Candidíase e Criptococose. Em ensaios de Dot Blot com gp43 purificada, foi comprovada a eficiência deste teste para o sorodiagnóstico da PCM em pacientes durante terapia antimicótica, apresentando alta sensibilidade e especificidade. Portanto, objetiva-se com este trabalho utilizar a técnica do ensaio de Dot Blot com a glicoproteína de 43 KDa como um método de diagnóstico para a PCM infecção em indivíduos expostos ao Paracoccidioides brasiliensis na área rural de Alfenas. A prevalência de positividade nos testes Dot Blot, ELISA e IDR foi 57,57% 56,71% 68,83% respectivamente. A proporção de positivos no teste de Dot Blot foi maior no gênero masculino. A maioria dos indivíduos positivos situa-se na faixa etária acima dos 30 anos. Os resultados mostram uma maior concordância entre o teste Dot Blot e ELISA. Devido à dificuldade na padronização no teste Dot Blot e a uma melhor reprodutibilidade e rapidez do teste de ELISA, os resultados sugerem que o teste de ELISA seja mais adequado para levantamento da prevalência da PCM em áreas rurais. / The paracoccidioidomycosis (PCM) is characterized as a deep mycosis and that affects mainly males and agricultural regions. Infection occurs by inhalation of fungal propagules in the environment, initially reaching the lungs and may spread to other organs. Early diagnosis of the disease prevents the formation of sequels and ensures that the worker gives continuity to their activities of daily living. The intradermal tests, immunodiffusion and ELISA are the most used in the diagnosis of PCM, however, may show cross-reactivity with other diseases such as histoplasmosis, candidiasis and cryptococcosis. Therefore, the objective of this study was to use the technique of Dot Blot assay glycoprotein of 43 kDa as a diagnostic method for PCM infection in individuals exposed to P. brasiliensis in rural Alfenas. Different variables were tested in the standardization of Dot Blot assay using sera samples from patient’s positive and negative individuals. The prevalence of positivity in Dot Blot, ELISA and IDR tests was 57.57%, 56.71% and 68.83% respectively. Seropositivity in the Dot Blot test was higher in males. Most positive individuals lie in the age group above 30 years. The results show a better agreement between the Dot Blot and ELISA. Due to the difficulty in standardizing the Dot blot test a better reproducibility and speed of ELISA, the results suggest that ELISA is more suitable for survey of the prevalence of PCM in rural areas.

Rotavírus têm sido identificados mundialmente como o mais importante agente etiológico de diarréias agudas não-bacterianas em animais jovens de várias espécies, incluindo a humana. Foi desenvolvido e avaliado um método de ELISA tipo “duplo-sanduíche” para a detecção de rotavírus a partir de material fecal. Para tanto, a amostra NCDV de rotavírus do grupo A foi propagada em cultivo celular com células MA-104. O vírus foi concentrado por ultracentrifugação e inoculado em coelhos e carneiros. Em seguida, as frações IgG, oriundas de amostras de soro dos animais, foram purificadas por cromatografia de troca iônica e absorvidas com soro total de ambas espécies animais, utilizando-se polímero de glutaraldeído, de modo a eliminar reações inespecíficas. A presença do rotavírus foi detectada pela IgG de carneiros e revelada pela IgG de coelho, usando como conjugado IgG de cabra anti-IgG de coelho conjugada à peroxidase. Os valores de diluição dos componentes do ELISA e o valor do ponto-de-corte foram definidos usando-se 26 amostras fecais (13 positivas e 13 negativas) de leitões, tendo como prova padrão a eletroforese em gel de poliacrilamida (PAGE). Aplicado a um painel constituído de 86 amostras fecais diarréicas de leitões, os resultados do ELISA foram: 100% de sensibilidade; 98,79% de especificidade, com uma concordância de 98,83%. A variância entre 86 repetições da mesma amostra foram 0,001 (para a amostra positiva) e 0,0002 (para a amostra negativa). Estes resultados demonstram que este ELISA é um teste sensível e específico para o diagnóstico de rotavírus a partir de material fecal. / Rotaviruses have been identified worldwide as a major etiologic agent of acute nonbacterial diarrhea in the young of many species, including humans. In this investigation was developed and evaluated a “double-sandwich” antibody ELISA method for detection of rotavirus from stool specimens. For that, the NCDV strain of rotavirus group A was serially cultivated in MA-104 cell culture. The virus was concentrated by ultra-centrifugation and inoculated in rabbits and sheeps. After that, the IgG of serum samples of the animals was purified by ion-exchange chromatography and absorbed with whole serum of both animal species using a glutaraldehyde polymer, in order to eliminate inespecific reactions. The presence of rotavirus was detected by the sheep’s IgG and revelated by the rabbit’s IgG, using a anti-rabbit IgG peroxidase conjugate developed in goat. The values of diluition of the components of the ELISA and the cut-off value were defined using 26 fecal samples (13 positive and 13 negative) of piglets. Following this procedure, the test was employed in a panel of 86 fecal samples from piglets with diarrhea, using as standard the polyacrilamide gel electrophoresis (PAGE) test. The results of the ELISA were: 100% of sensivity; 98.79% of specificity, with an agreement of 98.83%. The variance between 86 repetitions of the same sample were 0.001 (for one positive sample) and 0.0002 (for one negative sample). These results showed that this ELISA is an sensitive and specific screening test for rotavirus diagnosis from fecal material.

Thesis (M.Sc.( Medical Sciences)) --University of the North, 1983 / Refer to the document

The diagnosis of porcine circovirus-associated disease (PCVAD) in pigs requires the detection of characteristic clinical signs and pathological changes, and the detection of virus in tissues of affected pigs. To increase Australia’s capacity to independently diagnose PCVAD in Australia, techniques for the detection of Porcine circovirus type 2 (PCV2) infection in pigs were developed and are reported in this thesis. These techniques were applied to samples obtained from normal pigs and pigs with disease and confirmed the presence of PCV2 and PCVAD in the Australian pig herd. Viral DNA was detected in tissues of infected pigs by both standard PCR and real-time PCR techniques. The real-time PCR was more sensitive. While the conventional PCR was able to detect approximately 100 copies of the viral genome, the real-time PCR was able to detect 20 copies of the genome. An immunohistochemical (IHC) technique which was also developed enabled the visualisation of PCV2 antigen in fixed tissues of pigs with PCVAD. The techniques that were developed were applied to an examination of tissues from pigs affected by illthrift and increased weaner mortality in herds in South Australia, New South Wales and Western Australia. Lesions suggestive of the PCVAD postweaning multisystemic wasting syndrome (PMWS) were detected and virus antigen was detected in association with lesions. The nature of the clinical signs and histopathological lesions detected, coupled with the presence of PCV2 antigen, suggested that PCVAD was present in some Australian pig herds. Phylogenetic analysis of the strains of PCV2-associated with these disease outbreaks demonstrated they were of a type not previously detected in Australia and similar to strains associated with PMWS in North America. To further assist in investigation of PCV2 infections in the Australian pig herd, an enzyme-linked immunosorbent assay (ELISA) was developed that specifically detected antibody to PCV2 and not the related and non-pathogenic Porcine circovirus type 1. The development of this assay required the production of a virus capsid protein antigen using a prokaryotic protein production system. The ELISA was used to test serum samples form the Australian national pig serum bank. A high prevalence of PCV2 infection was detected in most pig herds examined in all Australian states. International trade in pig meat has resulted in many countries placing restrictions on the importation of pig meat, requiring imported pig meats to be cooked to destroy viral agents. This study investigated the in vitro resistance of an Australian strain of PCV2 to heat treatment at temperatures between 56°C and 85°C. The viability of the virus was determined by a combination of reverse transcriptase polymerase chain reaction (RT-PCR), and IHC to visualise viral capsid antigen within infected cells. This study indicated that PCV2 retained its infectivity following heating up to and including 75°C for 15 mins, but was inactivated following heating to 80°C and above. The investigations reported make a significant contribution to PCV2 research in Australia and ensure Australia’s capacity to independently investigate PCVAD in the Australian pig herd.

La síndrome de Sjögren és una malaltia autoimmune amb una prevalença força elevada a la població general (entre el 0,5% i el 3%). Tal com succeeix en d'altres malalties d'aquesta naturalesa, la presència d'anticossos antinuclears (ANA) està àmpliament acceptada com a prova important en el seu diagnòstic. En el caso de la síndrome de Sjögren, el principal marcador serològic és la presència d'anticossos dirigits contra l'antigen SS-B (La), tot i que en alguns casos també s'ha descrit la presència única d'anticossos dirigits contra l'antigen SS-A (Ro), format per dues proteïnes (Ro60 i Ro52) associades a diferents RNA citoplasmàtics.En el treball que es presenta, es descriu el procediment d'obtenció de les proteïnes antigèniques SS-B (La) i SS-A (Ro), per tal de poder ser utilitzades posteriorment en el desenvolupament de dues enzimoimmunoanàlisis (ELISA anti-SS-B (La) i ELISA anti-SS-A (Ro)) útils per a la detecció d'anticossos dirigits contra elles. L'estrategia que es va seguir fou la següent: es van preparar tres vectors d'expressió en Escherichia coli de les tres proteïnes (SS-B, Ro60 i Ro52) mitjançant la tecnologia del DNA recombinant, es van expressar les tres proteïnes recombinants en els microorganismes procariotes en forma de proteïnes de fusió, es van purificar les proteïnes per cromatografia d'afinitat, es van immobilitzar per adsorció passiva en microplaques de poliestirè, i es van optimitzar les diferents variables dels assajos de tipus ELISA. Els dos assajos desenvolupats es van avaluar mitjançant l'estudi de les constants diagnòstiques i analítiques i l'estudi de les característiques analítiques. Per a l'ELISA anti-SS-B (La), es van obtenir els següents resultats: sensibilitat diagnòstica del 96%, especificitat diagnòstica del 97%, coeficient de repetibilitat del 2,1%, coeficient de reproductibilitat del 10,0%, i absència de reaccions creuades i interferències. Per a l'ELISA anti-SS-A (Ro) els resultats foren els següents: sensibilitat diagnòstica del 88%, especificitat diagnòstica del 91%, coeficient de repetibilitat de l'1,9%, coeficient de reproductibilitat del 10,6%, i absència de reaccions creuades i interferències. / Sjögren's syndrome is an autoimmune disease with a high prevalence (between 0.5% and 3% of population). As other autoimmune diseases, the presence of antinuclear antibodies (ANA) in sera of Sjögren's syndrome patients is widely accepted as an important evidence for its diagnostic. The main serological marker of Sjörgren's syndrome is the presence of anti-SS-B (La) antibodies, although sometimes the presence of antibodies against the antigen SS-A (Ro) has also been described. The latter antigen is composed by two proteins (Ro60 and Ro52) binded to different cytoplasmatic RNA, so that antibodies against both proteins have been described in patient's sera.In that thesis, we describe the procedure for obtaining SS-B (La) and SS-A (Ro) proteins in order to be used as antigens in two enzymoimmunoassays (ELISA anti-SS-B (La) and ELISA anti-SS-A (Ro)) useful for the detection of antibodies against these proteins. We used the following strategy: first of all, three Escherichia coli expression vectors for the proteins (SS-B, Ro60 and Ro52) were constructed by means of recombinant DNA technology. The proteins were expressed in these prokaryote cells as fusion proteins, were purified by affinity cromatography and the proteins were immobilized by passive adsortion in polystyrene microplates. Finally, we optimized different variables of both ELISA and we evaluated the assays by calculating the diagnostic and analytical constants. The results obtained with the anti-SS-B (La) ELISA were the following: diagnostic sensibility 96%, diagnostic specificity 97%, intraserial CV 2.1%, interserial CV 10.0% and absence of cross-reactions and interferences. With the anti-SS-A (Ro) ELISA, the results obtained were: diagnostic sensibility 88%, diagnostic specificity 88%, intraday CV 1.9%, interday CV 10.6% and absence of cross-reactions and interferences.

Thèse de doctorat : Parasitologie : Paris 12 : 2005. / Thèse uniquement consultable au sein de l'Université Paris 12 (Intranet). Titre provenant de l'écran-titre. Bibliogr. : 192 réf.

Anoplocephala perfoliata is a species of parasitic worm that belongs to a group known as cestodes, which specifically target equine animals. As with all types of tapeworms, these parasites infect the gastrointestinal tract of their host, with devastating and potentially fatal consequences. The current lack of a sensitive and specific test for this parasite means that it continues to go undetected, hense this project aims to develop a novel and rapid diagnostic test with high sensitivity and specificity to help increase detection, thus precluding economic loss in the equine industry. The project details the development of three unique detection platforms; an ELISA, a lateral flow assay and an impedimetric immunosensor, aimed to detect IgA in saliva, since IgA is the dominant immunoglobulin of the mucosal immune system. IgA was therefore believed to be the ideal marker for rapid, specific and early indication of infection with A. perfoliata. Diagnosis using saliva samples was an integral part of this project, since it would allow for non-invasive sampling, by non-skilled personnel. A highly sensitive ELISA-based detection system was developed in this project for the detection of 3 different types of IgA. The first ELISA was developed to detect non-specific or ‘total’ IgA levels. Using a sandwich ELISA format, IgA was detectable with a LoD of ~0.04 ng/ml. A second ELISA was developed using the crude excretory/secretory (E/S) antigen, cultured from A. perfoliata worms, which were obtained by a vet during post-mortem examination of infected horses. The crude antigen mix was then used to fabricate an ELISA to detect specific IgA in saliva, produced against the E/S antigens. The crude antigen was then employed in a series of SDS PAGE and western blot experiments, which revealed the 12/13 kDa antigen as the main antigen detected by IgA in saliva. The 12/13 kDa was then electroeluted and used to immunise rabbits, in order to obtain anti-12/13 kDa antibodies, which were later used to purify large quantities of the 12/13 kDa antigen from the crude antigen mix. This allowed for the fabrication of the third and final ELISA, to detect IgA specific to the 12/13 kDa antigen. The 3 ELISAs were optimised throughout this project to ensure the most ideal conditions, such as antibody concentrations, sample dilutions, sample diluents, incubation temperatures and times were employed to obtain maximum assay sensitivity, specificity and productivity in a commercial setting. Testing samples (n = 24) using all 3 ELISAs and then standardising the specific IgA levels against the non-specific IgA, allowed for a novel and reliable detection method for A. perfoliata to be developed. This diagnostic test was developed in partnership with Austin Davis Biologics Ltd., who in April 2014 launched a screening programme which now offers horse owners an accurate means of testing their horses for A. perfoliata infections accurately. The second detection platform developed during this project was a lateral flow assay, whereby an immunochromographic strip was used to measure IgA levels in saliva. The studies performed determined the optimal conditions as using 40 µl of a 1:1,000 dilution of saliva using PBS(T) 1% as the sample diluent. The capture and control antibody were used at a concentration of 0.2 mg/ml, which were coated on the nitrocellulose membrane using an automated dispensing system (BioDot). The conjugate was labelled using gold nanoparticles, since it does not require any substrates or wash steps and its aggregation allows for immediate visual detection. A LoD of ~47 ng/ml was obtained for this assay. The final detection system investigated as part of this project was a label-less impedimetric immunosensor, whereby IgA was detected by means of electrochemical impedance spectroscopy (EIS). Polyaniline was the conductive polymer chosen to coat the surface of the screen printed carbon electrode, since the amine groups could be utilised to immobilise biotin molecules. A biotin-avidin complex was employed to ensure the uniform immobilisation of the capture antibody. Using the capture and control antibody at a concentration of 50 µg/ml and 10 mM ferri-ferrocyanide as the redox solution, IgA concentrations over a range of 100 – 0 ng/ml were investigated by Electrochemical Impedance Spectroscopy (EIS).