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There is considerable evidence indicating an association between aflatoxin ingestion and liver cancer in humans. The development of methods that would permit the monitoring of aflatoxin exposure in individuals would provide useful information in assessing human risk from this toxin. In this study, an ELISA technique, using a polyclonal antibody raised against aflatoxin B1 in the rabbit, was developed for monitoring the levels of aflatoxin excreted in human urine samples. Urine could not be used directly in the assay because high blanks were observed from the presence of some aflatoxin-like substances. A 'clean-up' procedure using Sep-Pak C[13] cartridges and immunoaffinity columns was developed. The methods were validated using [3]H-AFB[1] in both buffer and presumed uncontaminated human urine samples from Europeans. As AFB[1] itself is unlikely to be found in human samples, urine from marmoset monkeys treated with [14]C-AFB1 were also used in the validation processes as they are likely to contain a spectrum of aflatoxin metabolites similar to those in human urine. The levels of AFB[1]. equivalents were monitored by both radioactive counting and ELISA. The radioactive measurements demonstrated that the overall recovery of the ELISA method was approximately 50%. The failure to detect all of the aflatoxin contamination in the urine may be due to the inability of the antibody to detect most of the polar aflatoxin metabolites. When the ELISA method was used in monitoring aflatoxin excreted in patients with or without liver disease from Thailand, a range of aflatoxin levels in the urine samples were obtained. Since in Thailand, liver fluke infection, caused by Opisthorchis viverrini is a serious health problem in the area where there is high contamination with aflatoxin in food and a high incidence of liver cancer, the interaction between liver fluke infection and aflatoxin ingestion was also studied using hamster as an animal model. The results indicated that liver fluke infection may alter the pattern of aflatoxin metabolites excreted in hamster urine, which in turn could affect the results obtained from the ELISA. Measurements by the ELISA method of aflatoxin excreted in urine samples from Thai vegetarians; who are a population at high risk of exposure to aflatoxin suggested that this group of people excreted higher levels of aflatoxin, but a further study with a greater number of subjects would be necessary to confirm this finding.

Made available in DSpace on 2015-03-26T13:46:35Z (GMT). No. of bitstreams: 1 texto completo.pdf: 729175 bytes, checksum: d8be6fdfabfabf889bd81cc7f09142f9 (MD5) Previous issue date: 2008-05-16 / The Rhipicephalus (Boophilus) microplus is one of the most important arthropods in veterinary medicine due economic losses and health problems caused in cattle production, mainly in Central and South America, as well as in Australia. As an alternative to chemical control, immunization of bovines with Bm86 antigen to induce a protective immune response. A synthetic peptide, SBm 7462, derived from Bm86, has been shown great results in control of ticks. The construction and synthesis of one nucleotide sequence based on this peptide might be useful for design a DNA vaccine that has many advances than peptide vaccine. A gene, called seq1, was constructed with a three repetition of nucleotide sequence of SBm 7462. It was cloned into a pCIneo vector expression in mammals, transfected in VERO cells and injected in BALB/c mouse. Two methods were used to analysis of peptide expression in vitro: DOT ELISA and PAP. In both, the results showed that the nucleotide sequence (seq1) had not been express in VERO cells. In vivo, when mice were inoculated with the expression cassette they did not response in ELISA. They elevated antibody titles only when vaccinated with the syntetic peptide SBm 7462. And, the best titles of immunoglobulins were seen when the SBm 7462 was administered subcutaneously. After that, we insert a mutation at the begging of seq1, but, the sequenciament demonstrated that any initiation codon (ATG) had been inserted. / O Rhipicephalus (Boophilus) microplus é um dos mais importantes artrópodes em medicina veterinária devido perdas econômicas e problemas de saúde causados na produção de gado na América Central e do Sul, bem como na Austrália. Como alternativa ao controle químico, a imunização de bovinos com antígeno proteíco Bm86 induz uma resposta imune protetora. Um peptídeo sintético, SBm 7462, derivado da Bm86, tem obtido excelentes resultados no controle de carrapatos. A construção e síntese de uma sequência nucleotidíca baseada neste peptídeo podem ser útil para o desenho de uma vacina de DNA que tem muitas vantagens sob uma vacina sintética. Um gene, denominado seq1, foi construído repetindo três vezes a sequência nucleotidídica do SBm 7462. Ele foi clonado no vetor de expressão em mamíferos, pCIneo, transfectado em células VERO e injetado em camundongos BALB/c. Dois métodos foram usados para análise da expressão do peptídeo in vitro: DOT ELISA e PAP. Em ambos, os resultados demonstraram que a seqüência nucleotídica (seq1) não havia sido expressa em células VERO. In vivo, quando camundongos foram inoculados com o cassete de expressão eles não responderam ao ELISA. Eles elevaram os títulos de anticorpos, apenas, quando inoculados com o peptídeo sintético SBm 7462. Os melhores títulos de imunoglobulinas foram vistos quando o SBm 7462 foi administrado subcutâneamente. Após isso, inserimos uma mutação no início do seq 1, porém, o sequenciamento demonstrou que nenhum códon de iniciação (ATG) tinha sido inserido.

Made available in DSpace on 2016-08-29T15:37:23Z (GMT). No. of bitstreams: 1 tese_5652_CINTIA DAS CHAGAS BERNARDO.pdf: 612843 bytes, checksum: 51040d70cd6454b1e1355b49083aba94 (MD5) Previous issue date: 2012-02-24 / Métodos de diagnóstico da fasciolose hepática veem sendo estudados a fim de se propor técnicas mais acuradas, de fácil execução e de menor custo, que tenham aplicabilidade a campo. O objetivo do presente estudo foi validar kits comerciais® de ELISA para detecção de coproantígenos e anticorpos em soro e leite de bovinos infectados naturalmente por Fasciola hepatica. Numa primeira etapa, foram coletadas amostras de fezes, sangue e leite de bovinos naturalmente infectados por F. hepatica. Amostras de fezes de 577 animais foram processadas segundo a técnica coproparasitológica de sedimentação fecal para ovos de F. hepatica, e 92 amostras de soro e 43 de leite foram processadas segundo instruções do fabricante de um kit ELISA comercial®. Utilizou-se o Qui-quadrado de McNemar para comparação estatística, e calculou-se a sensibilidade e especificidade, valores preditivos e kappa dos kits®, sendo o exame coproparasitológico usado como padrão. Numa segunda etapa, foram avaliados ao abate 81 fígados bovinos dos quais 45 foram condenados por fasciolose. Foi realizada a contagem dos parasitos nos fígados condenados e coletada as amostras de fezes desses animais, além de 36 amostras fecais provenientes de animais que não tiveram os fígados condenados para nenhuma enfermidade. Das amostras de fezes foram separadas duas alíquotas sendo a primeira parte das amostras processadas pela técnica coproparasitológica de sedimentação e a outra, segundo instruções do fabricante de um kit ELISA comercial® para detecção de coproantígenos. Foram calculados os indicadores de validade e reprodutibilidade, e realizado o teste de correlação de Spearman e Qui-quadrado de McNemar, sendo utilizada como padrão ouro a condenação de fígados ao abate. Com os resultados obtidos nesses estudos, ficou claro que os kits comerciais® de ELISA apresentaram maior sensibilidade em relação ao exame coproparasitológico de sedimentação para o diagnóstico da fasciolose bovina, porém, para o diagnóstico da enfermidade a campo além da eficácia, deve-se levar em consideração também a operacionalização das técnicas, não descartando assim, o uso do exame coproparasitológico, sendo este menos trabalhoso e de menor custo em relação aos kits de ELISA testados.

Malaria är ett globalt hälsoproblem som orsakar många dödsfall runt om i världen varje år och nästan hälften av jordens befolkning ligger i riskzonen att drabbas av sjukdomen. I Indien drabbas mellan 2-3 miljoner människor varje år och det inträffar omkring 900 dödsfall. Malaria orsakas av Plasmodium sp. som är en protozoe, och det finns fyra olika arter som är patogena för människor, P. vivax, P. ovale, P. falciparium samt P. malariae. Vanliga metoder för att diagnostisera malaria är genom tunna och tjocka blodutstryk som färgas till exempel med Giemsa, Fields eller Leishmans färgningsteknik och studeras mikroskopiskt, Quantitative Buffy Coat (QBC), PCR tester, acridinorange färgning samt olika immunologiska tester för detektion av antikroppar eller antigen som till exempel enzyme-linked immunosorbent assays (ELISA) test och dipstick test. Syftet med denna studie är att utvärdera om en användning av SD Bio Line Malaria Antigen ELISA kit ger en mer känslig, tillförlitlig, praktisk samt mindre kostsam diagnostikmetod för malaria hos patienter med misstänkt malariainfektion än den nuvarande guldstandardmetoden, QBC tillsammans med blodutstryk, vid Christian Medical College and Hospital i Vellore. Patientproverna har i både ELISA testet samt QBC testet tillsammans med utstryk erhållit samma resultat vilket tyder på att SD Bio Line Malaria Antigen ELISA kitet skulle kunna vara en lika bra diagnostikmetod som QBC testet för diagnos av malaria. ELISA kitet har dock fler nackdelar, i jämförelse med QBC testet, så därför är slutsatsen att SD Bio Line Malaria Antigen ELISA kitet inte är en mer lämplig diagnostisk metod för malaria än den som används vid CMCH. Men då ELISA testet ändå ger en säker diagnos, enligt resultatet i studien, kan den vara ett lämpligt test inom något annat användningsområde.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Aneta Pražáková Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Determination of selected biomarkers of nephrotoxicity in urine and Plasma The discovery and development of novel biomarkers, that can be used for diagnosis of kidney damage earlier and more accurately, are needed for the effective prediction of drug-induced nephrotoxicity. Mechanisms of drug-induced nephrotoxicity include changes in glomerular hemodynamics, tubular cell cytotoxicity, inflammation, crystalline nephropathy, etc. Detection at initial stage of damage using sensitive and specific biomarkers belongs between one of the most important strategies in the treatment of acute kidney injury and renal failure. Although some these biomarkers do not show specificity and sensitivity, several promising biomarker candidates have been established recently to evaluate nephrotoxicity, e.g. selected KIM-1, cystatin C and NGAL. The advantages of these biomarkers compared to traditionally used biomarkers are higher sensitivity, specificity, just mentioned early diagnosis and non-invasiveness (the possibility of determination levels of the biomarkers from blood or urine). The aim of this diploma thesis was to determine...

Neospora has become a commonly recognized infectious cause of abortion in dairy cattle. The organism is associated with mid to late term abortion outbreaks with rates exceeding 30% in some herds. Cows infected with this organism exhibit no other clinical signs. While this disease has been reported in other parts of the country, no assessment of Neospora seroprevalence has been undertaken in the Southeast. This study sampled commercial dairy herds to assess Neospora seroprevalence in dairy cows and investigate its significance as an abortifacient agent in Virginia. Twenty four herds participated in the study. Twelve herds had DHIA reported annual abortion rates of 6% or greater (high abortion rate herds) and twelve herds had abortion rates of 2% or less (low abortion rate herds). High abortion rate herds were each paired to a low abortion rate herd (control) herd within the same county. A single blood sample was collected from all cows confirmed to be 90 to 240 days pregnant, with a maximum of thirty samples per herd. A random sample of cows was selected in herds with more than 30 pregnant cows between 90 and 240 days gestation. Neospora antibody titers were determined using a serum ELISA test at the California Veterinary Diagnostic Laboratory. Both mean and median seroprevalence of high and low abortion rate herds were compared using the Mann-Whitney Rank Sum test and the Median test, respectively. No significant difference was found in either case (p=0.56, p=0.41). These findings suggest that Neospora does not contribute significantly to the average abortion rate in Virginia's dairy cattle. / Master of Science

A lipoproteína de baixa densidade eletronegativa (LDL-) é um importante antígeno envolvido na patogênese da aterosclerose. A LDL(-) provoca resposta inflamatória e imunológica, levando à produção de autoanticorpos anti-LDL(-) e a formação subsequente de imunocomplexos (IC-LDL-), os quais também contribuem com o processo aterosclerótico. Diante disso, este trabalho teve como objetivo o desenvolvimento e validação de imunoensaios para quantificação plasmática de LDL(-), anti-LDL(-) e IC-LDL(-), assim como o desenvolvimento de ferramentas aplicáveis em um biossensor para LDL(-). Após a padronização de cada ELISA, foram avaliadas as características de desempenho dos métodos: limites de detecção (LD) e quantificação (LQ), precisão intra e inter-ensaios, exatidão, linearidade de diluição e interferentes. Os LD e LQ do ELISA para LDL(-) foram 0,423 mg/L e 0,517 mg/L de LDL(-), respectivamente. As concentrações plasmáticas de LDL(-) apresentaram linearidade quando os plasmas foram diluídos 1:1000, 1:2000, 1:4000 e 1:8000. Os LD e LQ do ELISA para anti-LDL(-) foram 0,0028 mg/L e 0,0032 mg/L de anti-LDL(-), respectivamente. Os plasmas apresentaram linearidade na diluição quando diluídos 1:100, 1:200, 1:400 e 1:800. Os LD e LQ do ELISA para IC-LDL(-) foram 0,023 g/L e 0,034 g/L de IC-LDL(-), respectivamente. Os plasmas apresentaram linearidade quando diluídos 1:12,5, 1:25, 1:50 e 1:100. Os três ELISAs apresentaram precisão intra e inter-ensaios e recuperação dentro dos limites requeridos para imunoensaios. Para o desenvolvimento de um biossensor para LDL(-), uma proteína recombinante, denominada GFP5-scFv, foi expressa em bactérias Escherichia coli da linhagem BL21(DE3). Para obtenção dessa proteína foi realizada a inserção da sequência de DNA de um fragmento variável de cadeia única (scFv) anti-LDL(-) em um vetor bacteriano com a sequência de DNA da proteína verde fluorescente (GFP5). Dessa forma, a GFP5-scFv é fluorescente e tem afinidade pela LDL(-). Também foram sintetizadas nanopartículas de ouro, as quais podem ser eficientemente utilizadas na supressão da emissão da fluorescência de GFP5-scFv. Portanto, os imunoensaios validados e os aplicativos desenvolvidos para o biossensor são ferramentas que tem potencial para serem utilizadas na avaliação da patogênese da aterosclerose. / The electronegative low-density lipoprotein (LDL) is an important antigen involved in the pathogenesis of atherosclerosis. The LDL(-) causes inflammatory and immune response, leading to production of autoantibodies anti-LDL(-) and the subsequent formation of immune complexes (IC-LDL), which also contribute to the atherosclerotic process. Thus, this study aimed to develop and validate immunoassays for quantification of plasma LDL(-), anti-LDL(-) and LDL-IC(-) as well as developing tools applicable to a biosensor for LDL(-). After the standardization of each ELISA, were evaluated the performance characteristics of methods: limits of detection (LD) and quantification (LQ), intra- and inter-assays precision, accuracy, linearity of dilution and interferences. The LD and LQ of LDL(-) ELISA were 0.423 mg/L and 0.517 mg/L of LDL(-), respectively. Plasmas showed linearity when diluted 1:1000, 1:2000, 1:4000 and 1:8000. The LD and LQ of anti-LDL(-) ELISA were 0.0028 mg/L and 0.0032 mg/L of anti-LDL(-), respectively. Plasmas showed linearity when diluted 1:100, 1:200, 1:400 and 1:800. The LD and LQ of IC-LDL(-) ELISA were 0.023 g/L and 0.034 g/L of LDL-IC(-), respectively. Plasma showed linearity when diluted 1:12.5, 1:25, 1:50 and 1:100. The three ELISAs showed good intra- and inter-assays precision and recovery within the limits required for immunoassays. To develop a biosensor for LDL(-), a recombinant protein, called GFP5-scFv was expressed in Escherichia coli BL21(DE3) strain. The obtainment of this protein was performed inserting the DNA sequence of an anti-LDL(-) single chain variable fragment (scFv) in a bacterial vector with a DNA sequence of green fluorescent protein (GFP5). Thus, the scFv-GFP5 is fluorescent and has affinity for LDL(-). Were also synthesized gold nanoparticles, which can be efficiently used for quenching the fluorescence emission of GFP5-scFv. Therefore, immunoassays validated and developed applications for the biosensor are tools that have potential to be used in evaluating the pathogenesis of atherosclerosis.