Rozprawy doktorskie na temat „Sandwich assays”

As the aging population is increasing worldwide, so is the prevalence of neurodegenerativediseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal dementia(FTD) and amyotrophic lateral sclerosis (ALS). Reliable biomarkers able to aid the diagnosis anddifferentiation of these diseases are needed in order to start the right treatment as early as possible.Due to its representative state of the central nervous system, cerebrospinal fluid (CSF) is afavorable sample material for biomarker discovery within neurodegenerative diseases. Alteredprotein levels of this body fluid might serve as a biomarker, but further validation of earlierfindings is needed. The aim of this project was to validate earlier studies suggesting potentialprotein biomarkers in CSF. From a list of 80 potential biomarkers in the CSF of patient samples,eight were chosen to be included in this validation effort. By utilizing a suspension bead array ina sandwich assay setup, 21 antibodies were tested in an initial screening. Antibody pairs that couldmeasure the protein levels in a dilution dependent manner was further optimized before individualpatient samples were analyzed. Sandwich assays targeting the three proteins Amphiphysin(AMPH), Chitotriosidase-1 (CHIT1) and Beta-synuclein (SNCB) were successfully developed andcorrelated to earlier generated data using a suspension bead array with a single binder setup.Therefore, the earlier findings of elevated levels of AMPH and SNCB in AD patients and CHIT1in ALS patients were successfully validated.
Prevalensen av neurodegenerativa sjukdomar såsom Alzheimers sjukdom (AD), Parkinsonssjukdom (PD), frontallobsdemens (FTD) och amyotrofisk lateralskleros (ALS) ökar i takt med denåldrande populationen. Pålitliga biomarkörer som kan hjälpa till vid diagnostiseringen av dessasjukdomar behövs för att starta rätt behandling så tidigt som möjligt. Ryggmärgsvätska, enkroppsvätska tillhörande det centrala nervsystemet, kan ge en inblick i det centrala nervsystemetstillstånd. Förändrade proteinnivåer i denna kroppsvätska skulle därför kunna fungera sombiomarkörer. Målet i detta projekt var att validera tidigare föreslagna proteinbiomarkörer iryggmärgsvätska. Utifrån en lista av 80 tidigare analyserade proteiner i ryggmärgsvätska hospatienter, inkluderades åtta proteiner i detta valideringsförsök. En antikroppsbaserad så kalladsandwich assay användes i en suspension bead array för att testa 21 stycken antikroppar i ett initialtscreeningsförsök. Antikroppspar som kunde mäta proteinnivåer på ett spädningsberoende vis i detinitiala screeningsförsöket optimerades vidare innan den utvecklade sandwich assayn användes föratt analysera proteinnivåer i individuella prover. Sandwich assays gentemot Amphiphysin(AMPH), Chitotriosidase-1 (CHIT1) och Beta-synuclein (SNCB) kunde bli framtagna ochkorrelerade gentemot tidigare genererat data från en single binder assay på ett framgångsrikt sätt.Projektet kunde därmed validera tidigare fynd som indikerat förhöjda nivåer av AMPH och SNCBi AD patienter, samt förhöjda nivåer av CHIT1 i ALS patienter.

La quantification de biomarqueurs spécifiques est un outil de diagnostic important. Les tests immunologiques standards tels que ELISA nécessitent de nombreuses étapes de lavage et une amplification du signal, en particulier à faible concentration. D'autre part, le transfert d'énergie résonant de type Förster (FRET) a été utilisé pour concevoir des tests biologiques homogènes en une seule étape qui ne nécessitent aucune étape de lavage, où le biomarqueur permet la formation d'un complexe "sandwich" impliquant des anticorps marqués par le donneur et d'autres marqués par l'accepteur. Le FRET du donneur vers l'accepteur fournit alors une signature optique de la formation du complexe, et donc du biomarqueur d'intérêt. Cependant, le FRET, qui est très sensible à la distance donneur-accepteur, ne se produit à un taux significatif que lorsque la distance donneur-accepteur est inférieure à 10 nm; la grande taille de nombreux complexes biologiques limite l'efficacité du transfert d'énergie, empêchant une détection sensible. Je propose ici une nouvelle modalité de transfert d'énergie qui utilise des microcavités optiques en solution. Ensuite, je décris un schéma de biodétection pour détecter un oligonucléotide biomarqueur de cancer en solution.À cette fin, j'ai conçu des structures de microcavité dans lesquelles des nanocristaux fluorescents sont placées à l'intérieur de microsphères diélectriques pour permettre un couplage fort de leur émission de fluorescence avec les modes de résonance de la cavité, appelés modes de galerie (WGM). J'ai étudié les propriétés structurelles et optiques de ces microcavités optiques. J'ai également caractérisé le transfert d'énergie entre ces modes et des nanoparticules acceptrices chargées de colorants présentes dans le champ évanescent, à quelques dizaines de nm au-dessus de la surface des microsphères. J’ai développé un modèle analytique pour caractériser les mécanismes de transfert d'énergie médié par les WGM (WGET). De plus, une comparaison entre WGET et FRET a révélé la supériorité du WGET dans le contexte de la construction de capteurs en termes de sensibilité et de portée de détection. Dans la dernière partie de la thèse, j’ai développé une stratégie pour fonctionnaliser ces microcavités optiques et leur permettre d'interagir avec des analytes cibles tels que l'ADN, l'ARN et les protéines avec une bonne spécificité. Cette stratégie a ensuite été adaptée pour fixer des sondes de capture d'ADN sur les microcavités activées par WGM. En utilisant les microsphères fixées à l'ADN comme donneur optique en combinaison avec des nanoparticules de colorants fonctionnalisées par un ADN complémentaire comme accepteurs optiques, un test de biodétection a été démontré avec succès pour détecter en solution un biomarqueur de cancer appelé survivine. Ce test a démontré une bonne sensibilité envers la cible, et s'est également avéré très spécifique. Le schéma de détection a été démontré dans un microscope confocal, au niveau de microsphères individuelles, puis transposé avec succès dans un instrument beaucoup plus simple tel qu'un spectrofluoromètre qui mesure la fluorescence de l'ensemble de la solution; la signature de la formation d'un complexe sandwich a été détectée efficacement.En conclusion, j'ai démontré que le transfert d'énergie assisté par microcavité présente plusieurs avantages par rapport aux tests FRET ordinaires. Un véritable test de biodétection basé sur le principe du WGET a également été conçu avec succès pour détecter des biomarqueurs du cancer avec une sensibilité et une spécificité élevées. Cette étude ouvre donc de nombreuses possibilités pour concevoir des tests plus performants et plus précis pour détecter diverses entités biologiques
Quantification of specific biomarkers is an important diagnostic tool. Standard immunoassays such as ELISA require extensive washing steps and signal amplification, in particular when the biomarker of interest is only present at very low concentrations. On the other hand, non-radiative Förster resonance energy transfer (FRET) has been used to design one-step homogenous bioassays which do not require any washing steps, where the biomarker enables the formation of a sandwich complex involving donor-labeled and acceptor-labeled antibodies. FRET from the donor to the acceptor then provides an optical signature of the complex formation, hence of the biomarker of interest. However, FRET which is highly sensitive to the donor-acceptor distance, only occurs in a significant rate when the distance between the donor and acceptor is less than 10 nanometers; thus the large size of many biological complexes limits the efficiency of energy transfer, preventing sensitive detection. Here I propose a novel energy transfer modality that uses solution-phase optical microcavities to enhance energy transfer. Following that, I describe a bio-sensing scheme designed to detect a cancer biomarker DNA in solution.To this aim, I have designed microcavity structures in which fluorescent colloidal quantum dots are located inside dielectric polymer microspheres to enable strong coupling of their fluorescence emission with the cavity resonance modes or whispering gallery modes (WGMs) of the microspheres. A detailed study was carried out to comprehend the structural and optical properties of these optical microcavities. I also characterized the energy transfer between these modes and acceptor dye-loaded nanoparticles present in the evanescent field, within a few tens of nanometers above the microsphere surface. An analytical model was constructed to provide insights into the WGM mediated energy transfer (WGET) mechanisms. Moreover, a comparison between WGET and FRET revealed the superiority of WGET in the context of building sensors with improved sensitivity and longer range of detection. In the last part of the thesis, a strategy is discussed in detail to provide biological functionalities to these optical microcavities which would enable them to interact with target analytes such as DNA, RNA, and proteins with high specificity, and moreover to reduce non-specific interactions. This strategy then was adapted to attach DNA capture probes onto the WGM enabled microcavities. Using the DNA attached microspheres as optical donor in combination with probe-DNA functionalized dye nanoparticles as optical acceptors, a biosensing assay has been successfully demonstrated to detect a cancer biomarker DNA called survivin in the solution phase. This assay did not only show good sensitivity towards the target, but also it has proven to be highly specific. The detection scheme has been demonstrated in a sophisticated confocal microscope at the single microsphere level, then successfully translated to a much simpler spectrofluorometer that measures fluorescence from the whole sample solution; the signature of the sandwich complex formation was also effectively detected.In conclusion, I demonstrated that microcavity-assisted energy transfer has several advantages over regular FRET assays. A real bio-sensing assay based on the WGET principle has also been successfully designed to detect cancer biomarkers with high sensitivity and specificity. This study thus opens up many possibilities to design high-performing and more accurate assays to detect varieties of biological entities

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
The present work describes the chemical study of leaves, stems and roots of the Tephrosia egregia Sandwith (Fabaceae) species, through the analysis of the volatile and nonvolatile constituent of the specie. The determination of the volatile chemical composition of leaves and stems of T. egregia showed the predominance of sesquiterpenes, with some monoterpenes and trisnor-sesquiterpenes. The comparison between identified constituents in the leaves (16; 88.75%) and stems (13; 85.22%) showed a similarity between the samples, although the trisnor-sesquiterpenes geijeren and pregeijeren were the major constituents in both the essential oils. From the chromatographic investigation of the essential oil of leaves, the trisnor-sesquiterpene dictamnol was isolated for the first time in the Tephrosia genus. The chromatographic investigation of the roots of T. egregia yields pongachalcone and praecansone B, of pongaflavone, of 6a,12a-dehydrorotenone and 12a-hydroxyrotenone, the mixture of sitosterol and stigmasterol, and their glucosylated forms, and the maackiain. From its leaves were isolated p-coumaric acid and ferulic acid. This two compounds had been isolated for the first time in the genus. The assays with extracts and isolated substances of T. egregia showed that the studied specie has very important larvicidal activity against Aedes aegypti and allelopathic activity, with best result for the ethanolic extract of the roots. In other hand, the bioassays of antimicrobial activity against of S. aureaus, E. coli, P. aeruginosa, S. choleraesuis and C. albicans, and the nematicidal activity on Meloidogyne incognta not shown significant results.
O presente trabalho descreve o estudo quÃmico das folhas, talos e raÃzes de Tephrosia egregia Sandwith (Fabaceae), atravÃs da anÃlise dos constituintes volÃteis e nÃo volÃteis da espÃcie. A determinaÃÃo da composiÃÃo quÃmica volÃtil das folhas e talos de T. egregia mostrou a predominÃncia de sesquiterpenos, com relatos de monoterpenos e trisnorsesquiterpenos. A comparaÃÃo entre os constituintes identificados nas folhas (16; 88,75%) e talos (13; 85,22%) revelou semelhanÃa entre os mesmos, onde os trisnor-sesquiterpenos geijereno e o pregeijereno foram os constituintes majoritÃrios em ambos os Ãleos essenciais. A partir do fracionamento cromatogrÃfico do Ãleo essencial das folhas foi isolado o trisnorsesquiterpeno dictamnol, relatado pela primeira vez no gÃnero Tephrosia. O estudo dos constituintes nÃo volÃteis foi iniciado a partir da obtenÃÃo dos extratos etanÃlicos das folhas, talos e raÃzes da espÃcie estudada. O fracionamento cromatogrÃfico das raÃzes levou ao isolamento das chalconas pongachalcona e praecansona B, da flavona pongaflavona, dos rotenoides 6a,12a-desidrorotenona e 12a-hidroxirotenona, da mistura dos esterÃides sitosterol e estigmasterol e de suas misturas nas formas glicosiladas e do pterocarpano maackiaina. A partir do extrato acetato de etila do decocto das folhas de T. egregia foram isolados os fenilpropanÃides Ãcido p-cumÃrico e Ãcido ferÃlico, descritos pela primeira vez no gÃnero estudado. Os ensaios de atividades biolÃgicas realizados para os extratos e substÃncias isoladas de T. egregia mostraram que a espÃcie estudada apresenta atividades larvicida sobre Aedes aegypti e alelopÃtica significativas, com destaque para o extrato etanolico das raÃzes (TERES). NÃo foram encontrados resultados significativos para os bioensaios de atividade antimicrobiana sobre cepas de S. aureaus, E. coli, P. aeruginosa, S. choleraesuis e C. albicans, assim como para atividade nematicida sobre Meloidogyne incognita.

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.

My dissertation hypothesis is that bovine trophoblast cells express cell-surface and secreted non-classical major histocompatibility complex class I (MHC-Ib) proteins which inhibit NK cells and other leukocytes by binding to inhibitory receptors (e.g., LILRB1, LILRB2, KIR2DL4, and/or CD94/NKG2A). Extremely polymorphic and ubiquitously expressed classical MHC class I (MHC-Ia) proteins, which present foreign antigenic peptides to CD8+ T lymphocytes, are involved in acceptance or rejection of tissue grafts. Non-classical MHC class I (MHC-Ib) glycoproteins, such as Human Leukocyte Antigen-G (HLA-G) and murine Qa-2, are important modulators of the maternal immune system during pregnancy. MHC-Ib proteins are: (a) oligomorphic or monomorphic, (b) expressed in specific tissues under specific condtions, and (c) produced as surface and/or soluble isoforms due to alternative splicing. Third trimester-bovine trophoblast cells express both MHC-Ia and MHC-Ib proteins. The MHC-Ib proteins expressed by trophoblast cells during the third trimester of pregnancy are encoded by four bovine leukocyte antigen (BoLA) loci: BoLA-NC1, BoLA-NC2, BoLA-NC3, and BoLA-NC4. Two MHC-Ia (N*01701 and N*01802) and three MHC-Ib (NC1*00501, NC3*00101 and NC4*00201) proteins showed cell-surface expression in transfection studies performed in murine P815 and human K562 cells. Two additional isoforms, NC1*00401 and NC2*00102, were not detected on the surface of these cells. Nevertheless, both class Ia proteins, N*01701 and N*01802, and five class Ib proteins, NC1*00401, NC1*00501, NC2*00102, NC3*00101, and NC4*00201, were detected in crude cell lysates on Western blots. Precipitation of proteins from culture supernatants showed that cell-surface MHC-Ia (N*01701 and N*01802) and MHC-Ib proteins (NC1*00501, NC3*00101, and NC4*00201) are shed from the surface of these cells into the media. The mechanism of shedding of these proteins is, however, not known. Monoclonal antibodies W6/32, IL-A88, H1A, H6A, H11A, H58A, and PT-85A recognized surface MHC-I isoforms with varying affinity. We were able to develop a sandwich enzyme-linked immunosorbent assay (ELISA) using either H1A or IL-A88 antibody as the capture antibody and the W6/32 antibody for detection. We produced monoclonal antibodies against cattle NC1*00501 and NC3*00101 proteins. One monoclonal antibody generated against BoLA-NC3*00101 was highly specific. Unfortunately, due to failure to clone the NC3*00101- hybridoma, we no longer have an infinite source of this monoclonal antibody for NC3*00101. We eluted peptides from NC3*00101-transfected MHC-null K562 cells and identified peptides using liquid chromatography-mass spectrum (LC-MS) analysis. Analysis of peptide binding data using the SAS Proc mixed statistical program, suggested that the peptide EVTNQLVVL is a potential peptide ligand, which can be used to make tetramers for enumeration of antigen-specific leukocytes.

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

The increased incidence of food pathogen outbreaks placed a new emphasis on the requirement of a rapid, sensitive, and reliable detection method for pathogens in food samples. Surface-enhanced Raman spectroscopy (SERS) is a technique that tremendously enhances the weak Raman scattering of an analyte by using a metallic nano-substrate. Herein, we developed an innovative SERS sandwich assay platform which is based on 3-mercaptophenylboronic acid (3-MPBA) or aptamer as a capturer, and 3-MPBA and silver nanoparticles (AgNPs) as the reporter for non-selective and selective detection of bacteria. Both optical and chemical (SERS mapping) imaging were used as mechanisms for bacterial detection and quantification. Using Salmonella enterica and Listeria monocytogenes as the model bacteria, we have identified a unique bacterial SERS signal upon the interaction between the captured bacteria, 3-MBPA and AgNPs, which was used as the base for reliable detection of bacteria using SERS mapping. The non-specific assay also possesses unique optical properties allowing for the enhanced visualization of bacteria at low microscope magnifications (10 and 20x objective lenses). Using 3-MBPA owe achieved sensitive detection and quantification of as low as 102 CFU/mL and a capture efficiency of 92.1% for nonselective detection of Salmonella. The capability of the assay method to detect specific bacteria using an aptamer was also demonstrated. Besides the SERS applications of this assay, it was discovered that the 3-MPBA coated gold chip developed for this assay enhances the visualization of bacteria under a light microscope allowing for facile and rapid detection and quantification. In anticipation for industrial applications, sample preparation methods and strategies were developed for simple and carbohydrate food matrices.

As demand for food increases, rapid testing methods are becoming increasingly important. In the past few years, yogurt has become popular. Yeast species are the most common spoilage organism, costing consumers and food companies money. A novel lateral flow assay has been developed to detect yeast oligonucleotide sequences. Gold nanoparticles were used as the standard reporter and fluorescent nanoparticles were developed as the novel reporter. The fluorescent nanoparticles were ruthenium-doped silica nanoparticles synthesized using the modified Stöber method. Visual analysis of assays using standard reporters showed the limit of detection to be 10 femtomoles of target sequence. Analysis of the fluorescent nanoparticles using a plate reader showed the limit of detection to be 0.027 femtomoles. The fluorescent reporter’s limit of detection is 1000 fold lower due to a sophisticated, more sensitive analysis method. Gold nanoparticles are appropriate for presence or absence testing, but fluorescent nanoparticles are best for obtaining quantitative data with low detection limits. Pathogens have been used as biological warfare for centuries. A brief review of common biowarfare agents is included. Yersinia pestis, the causative agent of the Plague, and Bacillus anthracis, the causative agent of Anthrax, are the focus. Additional work using gold nanoparticles as reporter in a sandwich assay is also included. The novel dye covered reporter was compared to the control, which was a single dye molecule linked to the reporter sequence. Repeated testing showed the novel reporter had a lower limit of detection and higher sensitivity due to increased ability to bind target.

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
國立清華大學
動力機械工程學系
103
The rapid spread of influenza-associated H1N1 viruses has caused serious concern in recent years. Therefore, there is an urgent need for the development of automatic, point-of-care devices for rapid diagnosis of the influenza virus. Conventional approaches suffer from several critical issues; notably, they are time-consuming, labor-intensive, and are characterized by low specificity. In this work, we present a new approach for fluorescence-based detection of the influenza A H1N1 virus using a sandwich-based aptamer assay that is automatically performed on an integrated microfluidic system. The entire detection process was shortened to 30 minutes using this chip-based system, and reagent consumption was decreased 5-fold in comparison to traditional methods. The limit of detection was significantly improved to 0.032 HAU due to the high affinity and high specificity of the H1N1-specific aptamers, suggesting that this microfluidic system could be useful in the detection of the H1N1 virus in situ.

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.

Thesis (M.S.)--Florida State University, 2004.
Advisor: Dr. 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 Apr. 18, 2005). Document formatted into pages; contains ix, 70 pages. Includes bibliographical references.

Yes
Ruthenium compounds have been shown to be promising alternatives to platinum(II) drugs. However, their clinical success depends on achieving mechanisms of action that overcome Pt-resistance mechanisms. Electron-deficient organoruthenium complexes are an understudied class of compounds that exhibit unusual reactivity in solution and might offer novel anticancer mechanisms of action. Here, we evaluate the in vitro and in vivo anticancer properties of the electron-deficient organoruthenium complex [(p-cymene)Ru(maleonitriledithiolate)]. This compound is found to be highly cytotoxic: 5 to 60 times more potent than cisplatin towards ovarian (A2780 and A2780cisR), colon (HCT116 p53+/+ and HCT116 p53−/−), and non-small cell lung H460 cancer cell lines. It shows no cross-resistance and is equally cytotoxic to both A2780 and A2780cisR cell lines. Furthermore, unlike cisplatin, the remarkable in vitro antiproliferative activity of this compound appears to be p53-independent. In vivo evaluation in the hollow-fibre assay across a panel of cancer cell types and subcutaneous H460 non-small cell lung cancer xenograft model hints at the activity of the complex. Although the impressive in vitro data are not fully corroborated by the in vivo follow-up, this work is the first preclinical study of electron-deficient half-sandwich complexes and highlights their promise as anticancer drug candidates.
UF150295/Royal Society; University of Bradford; Government Department of Business, Energy and Industrial Strategy; SBF003\1170/British Heart Foundation Springboard Award; AMS_/Academy of Medical Sciences/United Kingdom

abstract: Biological fluids, in particular blood plasma, provide a vital source of information on the state of human health. While specific detection of biomarker species can aid in disease diagnostics, the complexity of plasma makes analysis challenging. Despite the challenge of complex sample analysis, biomarker quantification has become a primary interest in biomedical analysis. Due to the extremely specific interaction between antibody and analyte, immunoassays are attractive for the analysis of these samples and have gained popularity since their initial introduction several decades ago. Current limitations to diagnostics through blood testing include long incubation times, interference from non-specific binding, and the requirement for specialized instrumentation and personnel. Optimizing the features of immunoassay for diagnostic testing and biomarker quantification would enable early and accurate detection of disease and afford rapid intervention, potentially improving patient outcomes. Improving the limit of quantitation for immunoassay has been the primary goal of many diverse experimental platforms. While the ability to accurately quantify low abundance species in a complex biological sample is of the utmost importance in diagnostic testing, models illustrating experimental limitations have relied on mathematical fittings, which cannot be directly related to finite analytical limits or fundamental relationships. By creating models based on the law of mass action, it is demonstrated that fundamental limitations are imposed by molecular shot noise, creating a finite statistical limitation to quantitative abilities. Regardless of sample volume, 131 molecules are necessary for quantitation to take place with acceptable levels of uncertainty. Understanding the fundamental limitations of the technique can aid in the design of immunoassay platforms, and assess progress toward the development of optimal diagnostic testing. A sandwich-type immunoassay was developed and tested on three separate human protein targets: myoglobin, heart-type fatty acid binding protein, and cardiac troponin I, achieving superior limits of quantitation approaching ultimate limitations. Furthermore, this approach is compatible with upstream sample separation methods, enabling the isolation of target molecules from a complex biological sample. Isolation of target species prior to analysis allows for the multiplex detection of biomarker panels in a microscale device, making the full optimization of immunoassay techniques possible for clinical diagnostics.
Dissertation/Thesis
Doctoral Dissertation Chemistry 2015

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.