Dissertations / Theses on the topic 'ELISA en sandwich'

Antecedentes: ICAM-1 y VCAM-1 pertenecen a un grupo de proteínas de tipo inmunoglobulina y se expresan sobre las células del endotelio vascular. La expresión de ICAMs es inducible e incrementa durante la activación celular, así el endotelio inflamado se caracteriza por un incremento en la expresión de ICAM-1 y de VCAM.1. El ensayo inmunoabsorbente ligado a enzimas (ELISA) utiliza una enzima como marcador para medir la formación de complejos antígeno-anticuerpo. El marcador enzimático que se emplea en estos análisis se conjuga con un ligando, que puede ser un antígeno, un anticuerpo específico para el antígeno de interés o un anticuerpo para el anticuerpo primario. La validación es la confirmación, mediante evidencia objetiva, de que se cumplen los requisitos de un método para la utilización o aplicación específica prevista. Objetivo: Determinar los niveles de moléculas de adhesión molecular I-CAM-1 y V-CAM-1 en pacientes; y realizar la validación del proceso mediante la técnica de ELISA Sándwich. Materiales y Métodos: Elaborar una curva de calibración para ICAM-1 y VCAM-1, medir muestras de pacientes aleatorios y determinar la muestra más alta, la cual será utilizada para realizar la validación, se llevan a cabo las pruebas de linealidad, repetibilidad y reproducibilidad. Resultados: Para la prueba de linealidad se obtuvieron los siguientes resultados: 1) VCAM-1 se obtuvo una r2= 0.99 dentro del intervalo 193 ng /mL a 1550.808 ng /mL, con un % de recobro entre 93-100 % 2) ICAM-1 se obtuvo una r2= 0.99, dentro del intervalo 160.173 ng /mL a 1256.808 ng /mL con un % de recobro entre 93-102 %. Para la prueba de repetibilidad 1) VCAM-1 se obtuvo un coeficiente de variación de 0.66 % - 2.73 %. 2) ICAM-1 se obtuvo un coeficiente de variación de 2.29 % - 4.86 %. En la prueba de reproducibilidad para 1) VCAM-1 se obtuvo un coeficiente de variación de 2.27 %, 2) ICAM-1 se obtuvo un coeficiente de variación de 3.90 %. Todas las pruebas son satisfactorias en ambos casos. Conclusiones: Dentro de los intervalos obtenidos en las pruebas de linealidad el método se comporta de manera lineal. De acuerdo con los resultados de repetibilidad y reproducibilidad podemos afirmar que el método es preciso. Dado el porcentaje de recuperación o recobro obtenido, podemos afirmar que el método es exacto. Los resultados obtenidos respecto a linealidad, precisión y exactitud, la técnica de Elisa Sándwich es adecuada para determinación de niveles séricos de ICAM-1 y VCAM-1.

Lyme borreliosis (LB) is caused by spirocheter of Borrelia burgdorferi sensu lato. There are different types of borrelia species and some differ in their ability to survive in the presence of the complement system. B. afzelii is complementresistant while B. garinii is complementsensitive. This is based on the ability to recruit immune regulators, such as factor H to the bacterial surface and prevent activation of the complement system. Some individuals may show anti-Borrelia antibodies without having developed clinical symptoms. This may indicate a more effective immune response against spirochetes. The aim of this study was to investigate differences in complement activation by measuring C3a and sC5b-9 with sandwich ELISA between two previously Borrelia-exposed groups; individuals with previous subclinical Lyme borreliosis (SB) and patients previously diagnosed with neuroborreliosis (NB), and a control group without signs of LB exposure. Samples analyzed in this study consisted of controls (Ctrl, n = 8st), SB (n = 60st) and NB (n = 22st). Plasma from the groups were activated with ACA1 and Lu59. To compare the relative increase between the groups, complement factor C3a and the soluble terminal complement complex, sC5b-9, were analyzed using sandwich-ELISA.The analysis of C3a and sC5b-9 showed higher activation with Lu59 than ACA1, which is consistent with previous studies. According to C3a-analysis, no significant differences were observed between the groups for neither ACA1 nor Lu59. According to sC5b-9-analysis, a significant difference between SB and Ctrl (p= 0,0081) for Lu59 was observed. Conclusion of the studie was that further studies are required to interpret how this complement activation affects LB from a clinical prespective.

La fasciolosis, producida por Fasciola hepatica, es una parasitosis cosmopolita de importancia médico-veterinaria que afecta a la ganadería lechera con prevalencias superiores al 80% que generan significativas pérdidas económicas. Por lo tanto, es importante contar con técnicas de diagnóstico que permitan la identificación temprana del parásito como las técnicas de captura cuando el antígeno se encuentra en baja concentración en la muestra. El objetivo del presente trabajo fue desarrollar un kit de ELISA para detectar antígenos de excreción-secreción de Fasciola hepatica. Se desarrolló un kit de ELISA tipo sandwich para la detección de antígenos de excreción-secreción de Fasciola hepatica, el cual alcanzó un límite de detección de 100 ng/mL, empleando una concentración óptima de anticuerpo de captura (anticuerpos policlonales de ratón) y segundo anticuerpo (anticuerpos policlonales de conejo) de 10 y 5 µg/mL, respectivamente; y una dilución óptima del conjugado (anticuerpo monoclonal anti-inmunoglobulinas de conejo conjugado con la enzima peroxidasa) de 1/1000. El kit de ELISA tipo sandwich, presento un valor de sensibilidad del 100%, una especificidad del 96.6% y valores predictivos positivos y negativos de 50% y 96.6% respectivamente; con relación al examen coproparasitológico directo. Al comparar el ELISA tipo sandwich con la contrainmunoelectroforesis (CIEF) se obtuvo una especificidad de 93.5% y un valor predictivo negativo del 100%. El kit de ELISA tipo sandwich desarrollado permite detectar antígenos metabólicos en muestras de heces de ganado ovino.
Tesis

Prostatacancer är den sjätte mest förekommande cancertypen i världen, och den tredje vanligaste cancertypen bland män. De olika typer av behandlingar som finns idag botar oftast inte sjukdomen. Det är därför viktigt att utveckla bättre behandlingsmetoder. Det är sedan tidigare känt att mangan kan orsaka apoptos i olika celltyper. Det ger en möjlighet att använda mangan för att hämma cancer och det är därför viktigt att veta vilka apoptotiska markörer som är involverade. Syftet med projektet var att undersöka om de sker en ökning av de apoptotiska markörerna kluven Kaspas-3 och kluven PARP efter manganbehandling av prostatacancerceller. Därefter kunna avgöra om manganbehandlingen har orsakat celldöd genom att inducera apoptos. Under projektet odlades prostatacancerceller (PC3) som sedan behandlades med 200 µM mangan under 6, 24 och 48 timmar. Därefter mättes mängden av proteinerna kluven Kaspas-3 och kluven PARP med hjälp av ett sandwich-ELISA kit. En tydlig stegvis ökning av apoptosmarkörerna med inkuberingstid hade förväntats. Det förväntade resultatet erhölls inte, för kluven Kaspase-3 ledde manganbehandlingen t.o.m. till en sänkning av koncentrationen. Det kan ha uppstått problem vid analysen som t.ex dålig lysering av cellerna eller ojämn tillväxt av dem. Det behövs fler studier för att utreda detta och för att undersöka andar potentiella apoptosmarkörer.
Prostate cancer is the sixth most common cancer type in the world, and the third most common cancer type among men. The different types of treatments that are available today do not usually cure the disease. It is therefore important to develop better treatment methods. It has previously been stated that manganese can cause apoptosis in different cell types. It provides an opportunity to use manganese to inhibit cancer and it is therefore important to know which apoptotic markers that are involved. The purpose of the project was to investigate whether an increase in the apoptotic markers cleaved Kaspas-3 and cleaved PARP after manganese treatment of prostate cancer cells. Thereafter, it can determine whether manganese treatment has caused cell death by inducing apoptosis. During the project prostate cancer cells (PC3-cells) were cultivated, then treated with 200 µM manganese for 6, 24 and 48 hours. The amount of the proteins cleaved Kaspas-3 and cleaved PARP was measured by using a sandwich ELISA kit. A clear gradual increase of apoptotic markers with incubation time was expected. The expected result was not obtained, for cleaved Kaspase-3 manganese treatment even decreased the concentrations. There may have been problems with the performance of the analysis such as poor lysis of the cells or uneven growth of the cells. More studies are required to investigate this and other potential apoptotic markers.

Slutsteget i immunförsvarets komplementaktivering innefattar en klyvning av komplementprotein C5, till C5a och C5b, vilket initierar bildandet av terminalt komplementkomplex (TCC) som i form av membran-attack-komplex (MAC) bildar cytotoxiska porer i bland annat gramnegativa bakterier. Bildandet av MAC kan blockeras av endogena regulatorer och TCC frisätts då som ett lösligt komplex, sC5b-9, i plasma. I examensarbetet studerades en variant av TCC, som i tidigare studier visats bildas oberoende av C3- och C5-konvertas när serum surgjorts till pH < 7,0 in vitro. Syftet med studien var att studera om detta atypiska TCC (aTCC) bildades hos grisar, som i en mekonium aspirationssyndrom (MAS)-modell, erhållit ett sänkt systemiskt pH in vivo. I syftet ingick också att etablera en ELISA-baserad metod för att analysera aTCC. I en sandwich ELISA användes monoklonal anti-C5a/C5a (desArg) (klon T13/9) som fångande antikropp och monoklonal anti-C9 (klon aE11) som detekterande antikropp för att analysera aTCC i plasmaprover från 18 MAS-grisar, samt i ett kontrollmaterial bestående av grisserum som surgjorts till pH 6,8 och 6,4 in vitro. Mängden aTCC i kontrollproverna ökade när pH sänktes men innehållet av aTCC i plasmaproverna minskade över MAS-studiens förlopp. När den relativa förändringen i aTCC relaterades till MAS-grisarnas slutliga pH kunde ett signifikant samband ses (p = 0,02) som visade att en större förändring i aTCC sammanföll med ett lägre slutligt pH. Nivåerna av aTCC var generellt sett högre i plasmaproverna jämfört med kontrollproverna vilket skulle kunnat bero på skillnader i plasma vs serum avseende aTCC eller att proverna kom från grisar med olika ålder och vikt. Avsaknad av grisspecifik standard och negativ kontroll samt lågt signal/brusförhållande bidrar till felkällor för analysen och denna kräver fortsatt optimering.
The late steps of complement activation involves a cleavage of complement protein C5, to C5a and C5b, which initiates the formation of terminal complement complex (TCC). The final complex is referred to as the membrane-attack-complex (MAC) which forms cytotoxic pores in, inter alia, gram-negative bacteria. The formation of MAC can be inhibited by endogenous regulators and the TCC is then released as a soluble complex, sC5b-9, in plasma. In the degree project, another type of TCC was studied, which in previous studies had shown to form independently of C3 and C5 convertases when serum was acidified to pH <7.0 in vitro. The purpose of the study was to investigate whether this atypical TCC (aTCC) was formed in piglets, which in a model of meconium aspiration syndrome (MAS), received a reduced systemic pH in vivo. The purpose was also to establish an ELISA for analyzing aTCC. Sandwich ELISA, with monoclonal anti-C5a / C5a (desArg) (clone T13/9) as a capture antibody and monoclonal anti-C9 (clone aE11) as a detection antibody, was used to analyse aTCC in plasma samples from 18 MAS piglets, and in control samples consisting of pig serum acidified to pH 6.8 and 6.4 in vitro. The amount of aTCC in the control samples increased when the pH was lowered, but the content of aTCC in the plasma samples decreased over the course of the MAS study. When the relative change in aTCC was related to the final pH of the MAS pigs, a significant relationship could be seen (p = 0.02) which showed that a major change in the aTCC coincided with a lower final pH. aTCC were generally higher in plasma samples compared with control samples, which could be due to differences in plasma vs serum for aTCC or that the samples came from pigs of different age and weight. Lack of pig-specific standard and negative control as well as low signal to noise ratio contribute to sources of error for the analysis and this requires continued optimization.

Mycobacterium tuberculosis provoque l'une des maladies qui présentent le taux de mortalité et de morbidité le plus élevé des Amériques et du monde. Dans les pays en développement, le diagnostic de la tuberculose (TB) repose sur la microscopie des frottis et des cultures bactériologiques. La première méthode a une faible sensibilité et la seconde met plusieurs semaines à atteindre un diagnostic de confirmation. L'absence de diagnostic rapide compromet les efforts de lutte contre la tuberculose, favorisant ainsi sa transmission à la population vulnérable. Actuellement, les nanoparticules magnétiques (MNP) fonctionnalisées par des biomolécules sont utilisées en biomédecine, en raison de leurs propriétés magnétiques, électriques et optiques. De cette manière, en appliquant des champs magnétiques externes, les MNP bio-fonctionnalisées sont utilisées pour détecter et concentrer les cellules et les biomolécules à partir d'échantillons biologiques.Dans ce travail, nous présentons la synthèse, la caractérisation et la bio-fonctionnalisation de nanoparticules magnétiques afin de développer un test ELISA en sandwich associé aux MNP, afin de détecter les antigènes de M. tuberculosis. À cette fin, des nanoparticules magnétiques ont été synthétisées par une méthode de co-précipitation. La surface des MNP a été amino-silanisée (MNP@Si@NH2) et caractérisée par des méthodes physico-chimiques.Les antigènes MTB évalués dans cette étude étaient: Hsp16.3, CFP10, ESAT6, MTC28, MPT64, protéine de 38 kDa, Ag85B et MoeX. Le clonage et l'expression de protéines recombinantes ont été réalisés dans le système de E. coli BL21 (DE3) pLysS. Des anticorps polyclonaux ont été produits chez des lapins Nouvelle-Zélande et des souris BALB/C, préalablement immunisés avec des antigènes recombinants purifiés. Des anticorps spécifiques (ab) ont été immobilisés sur les surfaces des MNP amino-silanisées. Les MNP@Si@ab ont été associées dans un test ELISA sandwich colorimétrique pour capturer et détecter les antigènes natifs du MTB à partir d’échantillons d’expectorations.La XRD, la spectroscopie Mössbauer, le potentiel zêta, la TEM et le FTIR ont validé l'obtention des MNP montrant un diamètre de cristal de diffraction de ~ 12,5 nm (10,48 ± 2,56 nm), une charge nette superficielle de +23,57 ± 2,87 mV, des profils caractéristiques de magnétite et une structure sphérique. De plus, une saturation en aimantation de 37,06 emu.g-1 a été observée. Pour la fonctionnalisation des surfaces de nanoparticules avec des anticorps, une méthode via l'utilisation d'ester activé (agent de couplage EDC/NHS) a été utilisée pour la formation de liaisons peptidiques. Des paramètres tels que le temps d'incubation, la concentration des agents de couplage et le niveau de saturation de surface des MNP amines silanisées (MNP@Si@NH2) ont déjà été standardisés.Enfin, des anticorps fonctionnalisés sur des MNP ont été utilisés pour capturer et détecter les antigènes recombinants et natifs de M. tuberculosis dans un test sandwich ELISA-MNP@Si@ab (dans un temps de réaction <4 h). Les antigènes ESAT6 et CFP10 ont été mieux différenciés dans les expectorations des patients atteints de tuberculose (fold value ~ 1,8). L'utilisation de MNP@Si@ab a amélioré la détection des antigènes du MTB dans des échantillons biologiques. Nos résultats sont encourageants, mais des évaluations supplémentaires sont nécessaire telles que la détermination de réactions croisées avec des échantillons d'expectorations provenant de patients atteints d'autres infections, la réalisation du test avec les expectorations fraîches de patients tuberculeux et la détermination de la sensibilité et de la spécificité de la méthode
Mycobacterium tuberculosis causes one of the diseases with the highest mortality and morbidity rate in the Americas and around the world. In developing countries, the diagnosis of tuberculosis (TB) is based on smear microscopy and bacteriological cultures. The first method has low sensitivity, and the second take several weeks to reach a confirmatory diagnosis. The lack of a rapid diagnosis compromises the efforts to control TB, favoring its transmission to the susceptible population. Currently, magnetic nanoparticles (MNPs) functionalized with biomolecules have been used in biomedicine, due the magnetic, electrical and optical properties. In this way, applying external magnetic fields, bio-functionalized MNPs is used to detect and concentrate cells and biomolecules from biological samples.In this work we present the synthesis, characterization and bio-functionalization of magnetic nanoparticles, to develop a sandwich ELISA assay associated to MNPs to detect antigens from M. tuberculosis. For this purpose, magnetic nanoparticles were synthesized by co-precipitation method. The MNP surface was amine-silanized (MNP@Si@NH2) and characterized by physical-chemical methods.The MTB antigens evaluated in this study were: Hsp16.3, CFP10, ESAT6, MTC28, MPT64, 38 kDa protein, Ag85B and MoeX. Cloning ad expression of recombinant proteins were made in E. coli BL21 (DE3) pLysS system. Polyclonal antibodies were produced in New Zealand rabbits and BALB/C mice, previously immunized with purified recombinant antigens. Specific antibodies (ab) were immobilized in the amine-silanized MNP surfaces. The MNP@Si@ab were associated in a colorimetric sandwich ELISA assay to capture and detect native MTB antigens from sputum samples.The XRD, Mössbauer spectroscopy, zeta potential, TEM and FTIR demonstrated the successful preparation of the MNPs showing a diffraction crystal diameter of ~12.5 nm (10.48 ± 2.56 nm), superficial net charge of ᶎ: +23.57 ± 2.87 mV, characteristic patterns of magnetite and a spherical structure. Additionally, a magnetization saturation of 37.06 emu.g-1 was observed. For the functionalization of nanoparticle surfaces with antibodies, active ester method (coupling agent EDC/NHS) were used for peptide bond formation. Parameters such as time of incubation, concentration of coupling agents and surface saturation level of amine-silanized MNPs (MNP@Si@NH2), were previously standardized.Finally, antibody functionalized on MNPs were used to capture and detect recombinant and native M. tuberculosis antigens in an ELISA-MNP@Si@ab sandwich test (in a reaction time <4 h). The ESAT6 and CFP10 antigens were better discriminated in sputum pooles from patients with TB (fold value ~ 1.8). The use of MNP@Si@ab improved the detection of MTB antigens in biological samples. Our results are encouraging, but the essay requires additional evaluations such as determining cross-reactions with sputum samples from patients with other infections, performing the test with fresh sputum of TB patients, and determining the sensitivity and specificity of the method
Mycobacterium tuberculosis causa una de las enfermedades con la tasa más alta de mortalidad y morbilidad en las Américas y en todo el mundo. En países en vías de desarrollo, el diagnóstico de tuberculosis (TB) se basa en microscopía de frotis y cultivos bacteriológicos. El primer método tiene baja sensibilidad y el segundo toma varias semanas para llegar a un diagnóstico confirmatorio. La falta de un diagnóstico rápido compromete los esfuerzos para controlar la TB, lo que favorece su transmisión a la población susceptible. Actualmente, las nanopartículas magnéticas (MNP) funcionalizadas con biomoléculas se han utilizado en biomedicina, debido a las propiedades magnéticas, eléctricas y ópticas. De esta manera, aplicando campos magnéticos externos, se utilizan MNP bio-funcionalizadas para detectar y concentrar células y biomoléculas a partir de muestras biológicas. En este trabajo presentamos la síntesis, caracterización y bio-funcionalización de las nanopartículas magnéticas para desarrollar un ensayo ELISA sándwich usando MNPs para detectar antígenos de M. tuberculosis. Para este propósito, las nanopartículas magnéticas fueron sintetizadas por el método de co-precipitación. La superficie de MNP fue amino-silanizada (MNP@Si@NH2) y se caracterizada por métodos físico y químicos. Los antígenos de MTB evaluados en este estudio fueron: Hsp16.3, CFP10, ESAT6, MTC28, MPT64, proteína de 38 kDa, Ag85B y MoeX. La clonación y la expresión de las proteínas recombinantes se realizaron en el sistema de E. coli BL21 (DE3) pLysS. Se produjeron anticuerpos policlonales en conejos de Nueva Zelanda y ratones BALB/C, inmunizados previamente con antígenos recombinantes purificados. Se inmovilizaron anticuerpos específicos (ab) en las superficies de MNP amino-silanizadas (MNP@Si@ab). El MNP@Si@ab fue utilizado en un ensayo ELISA sándwich colorimétrico para capturar y detectar antígenos de MTB nativos en muestras de esputo. La XRD, espectroscopia de Mössbauer, la potencial zeta, TEM y FTIR demostraron la preparación exitosa de los MNP, el cual mostró un diámetro de difracción del cristal de ~ 12.5 nm (10.48 ± 2.56 nm), carga neta superficial de ᶎ: +23.57 ± 2.87 mV, patrones característicos de magnetita y una estructura esférica. Además, una saturación de magnetización de 37.06 emu.g-1 fue observada. Para la funcionalización de superficies de nanopartículas con anticuerpos, se utilizó el método del éster activo para la formación de enlaces peptídicos. Parámetros tales como el tiempo de incubación, la concentración de los agentes de acoplamiento y el nivel de saturación de la superficie de las MNPs aminosilanizadas (MNP@Si@NH2) fueron estandarizadas. Finalmente, se usaron MNP funcionalizados con anticuerpos para capturar y detectar antígenos nativos y recombinantes de M. tuberculosis en una prueba sándwich de ELISA-MNP@Si@ab en un tiempo de reacción <4 h. Los antígenos ESAT6 y CFP10 se discriminaron mejor en las muestras de esputo de los pacientes con TB (fold value ~ 1,8). El uso de MNP@Si@ab mejoró la detección de antígenos de MTB en muestras biológicas con respecto a un sELISA convencional. Nuestros resultados son alentadores, pero el ensayo requiere evaluaciones adicionales, como determinar reacciones cruzadas con muestras de esputo de pacientes con otras infecciones, realizar la prueba con esputo frescos de pacientes con TB y determinar la sensibilidad y especificidad clînica del método

A importância em conhecer o padrão alimentar em mosquitos da Família Culicidae permite esclarecer alguns aspectos relacionados à transmissão de zoonoses e estimar o grau de contato humano-vetor que é fator relevante em estudos epidemiológicos. Com o objetivo de explorar o comportamento alimentar dessa Família, em área epizoótica de febre amarela silvestre, foram coletados exemplares nos municípios de Santo Antônio das Missões e Garruchos, Estado do Rio Grande do Sul. Fêmeas ingurgitadas foram obtidas por aspiração em ambiente de mata, no período de setembro de 2005 a abril de 2007 e identificadas segundo fonte de sangue ingerido através da técnica imunoenzimática ELISA de captura no sistema avidinabiotina. Foram testadas seis fontes de alimento: ave, bovino, eqüino, humano, macaco e rato. Os resultados obtidos mediante a padronização de anticorpos monoclonais possibilitaram demonstrar pela primeira vez o reconhecimento de sangue humano ingerido nesses mosquitos pelo emprego da subclasse IgG1 e comprovar a sensibilidade e especificidade da técnica ELISA de captura. No município de Santo Antônio das Missões, de um total de 190 amostras, 60,9% reagiram para sangue de boi, 23,6% para humano, 9,9% para ave, 1,9% para macaco e 3,7% para combinações de dois hospedeiros. Quanto às amostras referentes ao município de Garruchos, das 158 fêmeas capturadas na área Cachoeirinha pode-se observar reatividade para ave (16%), boi (29,6%), humano (36,8%), cavalo (4%), macaco (0,8%) e combinações de hospedeiros (12,8%), enquanto que para as 149 fêmeas pertencentes à área de São José, detectou-se sangue ingerido de boi em (51,5%), ave e humano (11,5%), macaco (6,2%), cavalo (0,8%) e mistos (18,5%). Aedes scapularis, Aedes crinifer, Culex (Culex) spp., Haemagogus leucocelaenus apresentaram maior número de fêmeas ingurgitadas nos dois municípios. Os resultados obtidos com Aedes scapularis sugerem ecletismo, conforme combinações detectadas em amostras de sangue de diferentes fontes. Haemagogus leucocelaenus apresentou a maior proporção de amostras contendo sangue humano em relação às demais fontes e essa característica traz implicações, por ser espécie incriminada na transmissão e por se tratar de área de ocorrência de epizootias de febre amarela.
The knowledge of mosquitoes Culicidae host feeding patterns permits to clarify some aspects related to zoonosis transmission and to estimate the degree of human-vector contact which is relevant in epidemiological studies. Aiming to explore the feeding behavior of these mosquitoes, specimens were collected in the municipalities of Santo Antônio das Missões and Garruchos, Rio Grande do Sul, an epizootic area of sylvatic yellow fever. Engorged females were collected by aspiration from forested areas from September 2005-April 2007 and their blood meals were identified using the avidin-biotin system of immunoenzymatic ELISA capture. Six blood meal sources were tested: bird, cattle, horse, human, monkey and rat. The result achieved with the species-specific IgG1 mAb was unprecedented for mosquito blood meal identification and reinforced the sensibility and specificity of the immunoenzymatic ELISA capture. Of the 190 samples from Santo Antônio das Missões, 60.9% reacted to cattle, 23.6% to human, 9.9% to bird, 1.9% to monkey and 3.7% to mixed blood meals. In Garruchos, of the 158 females collected in Cachoeirinha, 16.0% reacted to bird, 29.6% to cattle, 36.8% to human, 4.0% to horse, 0.8% to monkey and 12.8% to mixed blood, while of the 149 engorged females from São José, blood from cattle accounted for 51.5%, of blood identified, bird and human 11.5%, monkey 6.2%, horse 0.8% and mixed blood 18.5%. Blood engorged females of Aedes scapularis, Aedes crinifer, Culex (Culex) spp., Haemagogus leucocelaenus predominated in the two municipalities. The results obtained with Aedes scapularis suggests its eclecticism, according to the combinations of blood which were detected from different sources. Haemagogus leucocelaenus was found to have the highest proportion of samples containing human blood in comparison with other sources, which has implications, on account of being incriminated in the transmission and also for taking into consideration the outbreaks reported that underline the risk of yellow fever.

Les crevettes de grande taille de la famille des Penaeidae constituent le premier produit aquacole en valeur commerciale à l'échelle planétaire. Elles sont produites à 99% dans les pays en voie de développement de la ceinture sub-équatoriale. Parmi les causes limitant leur production, le WSS est l'agent pathogène viralayant provoqué le plus des pertes dans le monde. Notre étude a été orientée vers la reconnaissance des premiers stades de l'infection, susceptibles d'être la cible d'une action prophylactique. Nous avons testé l'utilisation d'une lignée cellulaire de poisson SSN-1 afin d'étudier les possibilités d'un développement in vitro. Seules des particules défectives ont été produites confirmant la haute spécificité des infections à virus de crustacés. Les recherches en microscopie électronique ont montré une similarité structurale du WSSV avec les virus (B, B2 et Baculo-B) des crabes. Ceci suggère que ces agents (B2 et WSSV) seraient tout deux de la famille des Nimaviridae et du genre Whispovirus. La comparaison avec le virus B2 permet de comprendre le rôle clef joué par la partie caudale de ces virus dans l'infection par son attachement à la membrane plasmique lors de l'infection chez son hôte spécifique
Penaeid family shrimp constitute the first aquaculture product in the world in terms of commercial value. They are produced in third world countries of the sub-equatorial belt. Among the causes limiting their production, one is the presence of the WSSV (White Spot Syndrome Virus), a pathogenic agent that produces massive mortality. Our aim was to investigate the first stages of the viral infection, in order to be used as target of prophylactic actions. A fish cell line (SSN-1) was used as model to tentatively develop in vitro studies. Only defective particles were produced confirming the high specificity to crustacea of the infection with crustacean virus. Electron microscopy showed structural similarities between the WSSV and B, B2 and Baculo-B viruses of crabs. This suggests B2 may belong also to the family Nimaviridae, genus Whispovirus. This comparison with B2 virus gives the possibility to understand the role played by the tail-like extension of these viruses in the infectious process by attachment to the plasmic membrane at the beginning of the infection in its specific host

碩士
高雄醫學大學
生物醫學暨環境生物學研究所
101
Food and Drug Administration (FDA, U.S.A.) has approved the PEGylated technology that can use in clinical treatment. With the times, numbers of PEGylated molecule has entered to clinical use, such as protein, liposome and small molecule. In previous study, we have established an anti-PEG sandwich ELISA (AGP4/3-3-Biotin) that can quantify the pegylated compounds. However, the sensitivity of this PEG quantification platform might be affected that depend on the different kind of the pegylated molecules. In this study, we established an anti-PEG sandwich ELISA according to the different combination of the high affinity anti-PEG antibodies that can extensive quantification to any kind of pegylated compound. We also established the cell line (293T/anti-PEG) that can anchor the anti-PEG antibody on mammalian cell membrane. These high affinity anti-PEG antibodies and the anti-PEG cell line can be used in sandwich ELISA to quantify pegylated molecules. These anti-PEG sandwich ELISA provide a high sensitive, convenient and inexpensive tool to the pharmacokinetic studies of PEGylated molecules.

Thesis (M. A.)--Florida State University, 2006.
Advisor: Yun-Hwa Peggy Hsieh, Florida State University, College of Human Sciences , Dept. of Nutrition, Food, and Exercise Science. Title and description from dissertation home page (viewed Jan. 2, 2007). Document formatted into pages; contains xi, 115 pages. Includes bibliographical references.

Thesis (M.S.)--Florida State University, 2006.
Advisor: Yun-Hwa Peggy Hsieh, Florida State University, College of Human Sciences, Dept. of Nutrition, Food and Exercise Sciences. Title and description from dissertation home page (viewed Sept. 19, 2006). Document formatted into pages; contains viii, 75 pages. Includes bibliographical references.

碩士
國立交通大學
機械工程系所
102
The goal of this research is to optimize sandwich enzyme-linked immunosorbent assay (ELISA) in microarray format with 4 different proteins (TNFα, α1-Antitrypsin, Cystatin C and E-Cadherin), which can potentially be used as biomarkers for screening early stage of diabetic nephropathy (DN). The specific objectives are to: 1. optimize assay parameters with Taguchi Method and 2. systematically evaluate the effectiveness of incorporating Tyramide Signal Amplification (TSA) in an antibody microarray. Two optimization rounds based on Taguchi method were performed in both assay with and without TSA. In the first round, for assay without TSA, we tested four different factors including capture antibody (CapAb), analyte, detection antibody (DetAb) and streptavidin-conjugated Cy5 (SA-Cy5) at three different concentrations. For assay with TSA, we tested one additional factor, streptavidin-conjugated horseradish peroxidase (SA-HRP). In the second round, for assay without TSA, we tested five factors including four different incubation steps (blocking, analyte, DetAb, SA-Cy5) and SA-Cy5 concentration at two different levels. For assay with TSA, additional tyramide incubation time was also tested. For each factor, the level with highest S/N ratio in response table was selected as the optimum condition. After Taguchi optimization, the limit of detection (LOD) for 4 different analytes were improved by 16.9% - 89.7% and 24.1% - 79.0% for assay without and with TSA respectively. Our result also verifies the effectiveness of incorporating TSA to improve the LOD, which will be beneficial for detection of low concentration analytes.

碩士
國立宜蘭大學
生物技術與動物科學系
104
Porcine circovirus-associated disease (PCVAD) is an important disease that affects the pig industry, and the major pathogen is Porcine Circovirus type 2 (PCV2). In order to cope with the heavy loss caused by PCV2, leading pharmaceutical and biotech companies have launched PCV2 vaccines and diagnostic reagents. Currently on the market, the diagnostic products for PCV2 detection aim to detect anti-PCV2 antibodies in sera. No commercial products that detect the antigen of PCV2 virus particle are available. The object of this dissertation was to develop an enzyme-linked immunosorbent assay (ELISA) to detect antigens of PCV2. Firstly, monoclonal antibodies against PCV2 virus particle were produced. A large amount of virus obtained from cell culture was purified and used to immunize mice as well as produce hybridomas. After several screenings, a hybridoma cell line, 3A10-D1-H2, capable of secreting antibodies against PCV2 virus particle was successfully isolated. The monoclonal antibodies were verified to recognize PCV2 virus particle by ELISA, indirect immunofluorescence assay (IFA), etc. Purified monoclonal antibodies obtained from murine ascites and rabbit polyclonal antibodies were used to develop an ELISA. Results showed that the sandwich ELISA successfully detected PCV2 viruses produced via cell culture and the sensitivity for purified virus reached ng/ml levels. On the other hand, the monoclonal antibodies produced from this study have the ability to neutralize PCV2 virus infection. The sequence of the high viability zone of PCV2 antibody was also revealed by nucleotide sequence analysis with the hybridoma cell line. The information paves the way for subsequent development of therapeutic chimeric antibodies.

碩士
國立中興大學
食品暨應用生物科技學系所
107
Peanut is a common food ingredient and also is a one of the most allergenic food. The symptoms of peanut allergy includes skin redness, itching, abdominal pain. In more severe cases, anaphylaxis sets in, leading to breathing trouble and even death. Peanut allergic reactions can be induced by less than 1 peanut kernel. Different processing methods could alter food allergenicity, i.e. increasing or decreasing IgE reactivity. Therefore, the aim of the study was to develop an sandwich ELISA for the detection and quantification of major peanut allergen Ara h 1 in processed foods. The enzyme-linked immunosorbent assay (ELISA) is a easy and low-cost analytical method that provide a good specificity and sensitivity. Ara h 1 is the most abundant of the peanut seed proteins, and resistant to heat and digestion, thereby preserving allergenicity of peanuts during food processing. In this study, we have established a sandwich ELISA based on polyclonal antibody (pAb) to measure the content of the major peanut allergen Ara h 1 in foods. The optimal concentrations of the capture antibody and detection antibody were 12.5 µg/mL (37 oC, 60 min) and 1 µg/mL (37 oC, 45 min) , respectively, and blocking with 5% glycine (4 oC, overnight), samples were incubated for 90 min at 37 oC, and SA-HRP was used at a concentration of 150 ng/mL. The limit of detection is 0.029 μg/ml Ara h 1 in PBS, and The standard curve was y = 1.44595 ln(x) + 7.22775 (R2 = 0.97412). This assay was used to detect Ara h 1 in chocolate, cookie and milk products, and the detection limit were 10.82, 6.86 and 35.40 μg/ml, respectively. Matrix effects can be observed, and the milk matrix severely affected the measurements. This assay could detect Ara h 1 in many kinds of food smples.

碩士
國立中興大學
微生物暨公共衛生學研究所
100
Classical swine fever (CSF) caused by clsaaical swine fever virus (CSFV) is a high mortality disease in pigs. CSFV belongs to the genus Pestivirus of the Flaviviridae family, and infected pigs are often characterized by fever, hemorrhages and leucopenia on pathogenic findings. Glycoprotein Erns of CSFV contains an unique RNase functional domain and could induce humoral immune response, that plays an important role in the pathogenesis. The purpose of this study is to prepare the anti-CSFV Erns monoclonal antibody (Mab) for further applying as a diagnostic tool. Three Mabs 1-41, 3-8 and 10-12 specific to recognize yeast or E. coli expressed Erns were obtained and all were determined to be the type of IgG1 subclass. The antigenic sites recognized by Mabs 1-41, 3-8 and 10-12 were mapped using E. coli subunit and peptide scanning. The result shows to recognize the Erns region of amino acid residues 161-190, 151-170 and 141-150, respectively. Furthermore, the Mab 10-12 was used as a capture antibody to develop a yeast-expressed Erns subunit (yErns/N190) based indirect sandwich ELISA for detecting swine antibody to Erns. The assay demonstrated a high sensitivity of 97% (28/29) and a moderate specificity of 85% (22/26) when compared with a commercial CHEKIT Classical Swine Fever Marker Test (IDEXX) Erns blocking ELISA. This diagnosis tool could detect large numbers serum with easy manipulation and low cost.

by Michael Yiu-kwong Leung.
Thesis (Ph.D.)--Chinese University of Hong Kong, 1998.
Includes bibliographical references (p. 158-168).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) with a monoclonal antibody was developed and evaluated for the detection of prune dwarf ilarvirus (PDV) in sweet cherry trees {Prunus avium). A reverse transcribed polymerase chain reaction test was also developed to establish the incidence of PDV in 40 sweet cherry trees and to confirm the absence of virus in 15 control trees. Trees with two-thirds of their leaves positive for PDV by TAS-ELISA would be identified with 99% probability by testing four leaves per tree. The monoclonal antibody did not cross- react with Prunus necrotic ringspot ilarvirus in the TASELISA. The nucleotide sequence of PDV RNA1 was determined. The RNA consists of 3374 nucleotides and encodes a single open reading frame of 3168 nucleotides. The putative translation product is 1055 amino acids in length with a calculated molecular mass of 118.9 kDa. Both the nucleic acid and the translated amino acid sequences show stronger homology to RNA1 and the corresponding translation product (ORF1) of alfalfa mosaic alfamovirus (AMV) than' to citrus leaf rugose ilarvirus, the only other ilarvirus for which RNA1 sequence data is available. There is extensive sequence homology in the 3'-untranslated regions of PDV RNA1 and the 3'-regions of other ilarvirus and AMV RNAs. The reported sequence and its single open reading frame conform to the genomic organization typical of the Bromoviridae genus. Clones representing sequence from the 5'and 3'-end of RNA1 were used to construct a deletion-type defective interfering particle and its ability to replicate in vivo was assessed.

碩士
國立臺灣大學
獸醫學研究所
94
Avian polyomavirus (APV) infection and psittacine beak and feather disease (PBFD) are the most common viral diseases of psittacine birds. APV was first isolated from budgerigars in the early 1980s. APV belongs to genes Avipolyomavirus of the family Papovaviridae and causes acute fatal disease in young budgerigars with 100% mortality rate. APV infection is also known as budgerigar fledgling disease. PBFDV, categorized into genes Circovirus of the family Circoviridae, affects over 60 species of wild and captive psittacine birds. In this study polymerase chain reaction (PCR) was applied to diagnosis either APV or PBFDV infections in psittacine birds of Taiwan. From 2002 to 2005, the positive rate of APV, PBFDV, and APV/PBFDV infection over 165 cases were 15.2%, 41.2%, and 10.3% respectively. APVs indicated over 97% nucleotide identity in VP1 and T antigen coding regions. PBFDVs had over 92.2% and 83.3% nucleotide identity in ORF V1 and ORF C1 sequences. Another aim of this study is to prepare Sandwich ELISA for detection of APV and to analyze the specificity and sensitivity of Sandwich ELISA. First of all, we applied prokaryotic (E. coli.) system to express the viral structural protein VP1. The expressed VP1 were specifically recognized by his-tag and anti-APV antibodies. The recombinant protein was used as an immunogen to inject BALB/c mice for preparation of hybridomas which produced anti-VP1 antibodies. Eight monoclonal antibodies secreting hybridomas were obtained as determined by ELISA and Western blot. After screening of these 8 monoclonal antibodies, one monoclonal antibody, D10-6, is used to develop the Sandwich ELISA for the detection of APV. No significant difference was formed between PCR and Sandwich ELISA at the sensitivity. The Sandwich ELISA could be applied for diagnosis of APV infected budgerigars, and could further be applied to immunoassay strip for the rapid diagnosis of APV infection in budgerigars.

碩士
國立中興大學
畜產學系
87
捌、英文摘要 Development of nenotal kid’s passive amd active immunity and the assay of sandwich ELISA applied for the measurement of goat’s immunoglobulin G Waun-Pin Wang Abstract Milk immunoglobulin G (IgG) is main source of immunoglobulin and originates from the blood of ruminant dam. During parturition, the levels of mother blood and milk IgG are distinctly elevated. The newborn kids have low levels of IgG and low titer of innate immunity because the blood IgG of dam is not cross the placenta of dam side. In addition, the gut closure appears within 1-2 day after birth. The colostral IgG is acquired for passive immunity in kids. The present study was conducted to test the ability of passive immunity after kids fed colostrum, heat inactivated colostrum, milk of late lactation or cRBC-induced colostrum. Futhermore, the alterations of serum and colostral IgG in prepartum and postpartum of dams were examined. The bleeding and collecting mammary secretes of dams were at prepartum (-7,-3,-1day), parturition (0day), and postpartum (1,3,7day) respectively. Two dams were immunized with cRBC (s.c) once every week before 4 weeks of parturition. Their kids were fed cRBC-induced colostrum after birth. Normal kids were randomly assigned to feed colostrum, heat inactivated colostrum or milk every 6hr after birth and were bled prior to feeding for 5 consecutive days. The kids were administrated with chicken RBC at 3 and 5 weeks old respectively and were bled once at 6 weeks old. After electrophoresis and electroelution, the purified goat serum and colostral (IgG, IgG1, IgG2) were higher purity. By using this purified IgG as a immunogen to immunize the rabbit, the polyclonal antibodies were characterized and applied to measure the serum and colostral IgG contents. The SDS-PAGE and western blot showed that the highest level of serum IgG was at 18 to 24 hr after birth .The serum IgG was the lowest level in the kids fed milk of late gestation. The results imply that the passive immunity or absorption of kid increases after colostrum feeding. The IgG concentration of jugular and mammary veins in dams showed biphasic patterns during 7day prepartum to 1 day postpartum and 3-7 day postpartum. At postpartum, the milk IgG level of dams showed a peak at parturition. At postpartum, the milk IgG level of dams showed a peak at parturition. At postpartum, the milk IgG level of dams was dramatically declined. The titer of anti-chicken RBC was not significant difference between kids fed colostrum, heat inactivated colostrum or milk of late gestation at 3 and 6 weeks old. These results suggest that the active immunity of kids may not be affected by the ammounts of Ig in feeding milk. The anti-chicken RBC titers were significantly higher in jugular vein and mammary secretes of cRBC-induced dam at day 1 postpartum was higher titer (P<0.05) of anti-chicken RBC than that non cRBC-induced dam. In addition, this trend was decreased at day 2 to day 7 postparturition. Comparing to the commercial bovine serum IgG1 and IgG2, the relative amounts of goat serum and colostral (IgG, IgG1, IgG2) were determined by sandwich enzyme link immunosorbent assay.