The purpose of this study is to demonstrate the ability of surface enhanced Raman scattering (SERS) active particles to enable multiplexed immunoassays in a lateral flow format for point of care (POC) testing. The SERS particles used for this study are chemically active glass coated gold particles, containing tracer molecules which in principle can be chosen to provide Raman Spectra with unique features allowing multiple tracers to be simultaneously measured and distinguished without interference between each other. Lateral flow immunoassay technology is the important part of this study and can be conveniently packaged for the use of other than highly skilled technicians outside of the laboratory. A well-known (single channel - simplex) device for the pregnancy test is a typical example of the lateral flow assay. Similar formats have been/are being developed by others for a range of POC applications – but most diagnostic applications require simultaneous determination of a range of biomarkers and multiplexed assays are difficult to achieve without significant interference between the individual assays. This is where SERS particles may provide some advantages over existing techniques. Cardiac markers are the growing market for point of care technology therefore biomarkers of cardiac injury (Troponin, myoglobin and CRP) have been chosen as a model. The object of the study is to establish the proof of concept multiplexing assay using these chosen biomarkers. Thus, initially all different particles were characterised in single and mixture form. Also development of conjugate chemistry between antibodies for each analyte that have been purchased from commercial sources and SERS particles were analysed using different conditions like buffer, pH and antibody loading concentration to get the optimum intensity. The selected SERS particles and their conjugates were tested for size, aggregation and immune quality using a range of techniques: ultraviolet-visible (UV/Vis) absorption spectroscopy, dynamic light scattering (DLS) and lateral flow assay. These characterisations methodologies gave the understanding of optimum conditions of the each conjugates and individual’s behaviour in mixture conditions as well. After the characterisation all conjugates were tested singularly on the lateral flow assay using buffers and serum. The results of this single analyte immunoassay explained the individual’s bioactivity on the lateral flow strip. Further in study, multiplex assay have been demonstrated in serum. These outcomes have described each candidate characteristic in a mixture form on the lateral flow strip. In order to get the optimum Raman intensity from multiplex assay, the detection and capture antibodies loading concentrations were tuned in the assay. Later on different combinations (high, medium and low concentrations) of all three analytes were analysed and has found some interferences in multiplex assay. To investigate these issues various aspect were considered. First of all, different possibilities of non-specific interactions between the co-analytes and antibodies were tested. In addition, steric hindrance and optical interference investigations were performed via several assays and analysis using Scanning electron microscopy. The outcomes have confirmed related optical interferences. Therefore other assay (wound biomarkers) established to eliminate the interferences. In summary, the works reported here have built and test the equipment and necessary reagents for individual assays before moving on the more complicated task. In addition, the entire study has given a deep knowledge of multiplex assay on a single test line including the investigation of the issues for selected cardiac biomarkers and their applications in the future.

Development of chimeric, recombinant peptides, proteins and enzymes expands the availability of protein/enzyme–based tools for cellular analysis and new assay platforms. Ideal protein reagents for cellular analysis must translocate into a variety of cells with minimum cell damage, retain stability and biological activity within the cell during analysis, and provide a reliable, measurable signal. This work focused on development, characterization and utilization of chimeric recombinant peptide, protein and enzyme reagents for cellular analysis and immunoassays. A cell-penetrating, fluorescent protein substrate (PKAS) was developed to monitor intracellular protein kinase A activity in cells without the need for cellular transfection. PKAS translocated into HeLa cells, βTC-3 cells and pancreatic islets with minimal toxicity. Upon cellular loading, glucose dependent phosphorylation of PKAS was observed in both βTC-3 and pancreatic islets via capillary zone electrophoresis. In pancreatic islets, maximal PKAS phosphorylation (83 ± 6 %) was observed at 12 mM glucose, whereas maximal PKAS phosphorylation (86 ± 4 %) in βTC-3 cells was with 3 mM glucose indicating a left-shifted glucose sensitivity. A cell-penetrating luciferase chimera (Luc-TAT) and a cell-penetrating phospholipid nanoshell entrapped luciferase (Luc-PPN) was constructed to monitor dynamic changes in intracellular ATP levels in mammalian cells. Upon cellular loading, the activity of Luc-TAT and Luc-PPN was monitored with time. Luc-TAT lost approximately 50% activity within one hour, and decreased rapidly over time. In contrast Luc-PPNs retain approximately 95% activity in 1 hour and 77% after 12 hours showing longer biological lifetime. Luc-PPNs were able to detect dynamic ATP changes in intact HeLa cells in the presence of KCN and NaN3. The bioluminescence returned to background levels within 8-10 minutes after treatment with KCN, whereas NaN₃ showed ~ 40% reduction. Two novel recombinant human parathyroid hormone (hPTH) analogs hPTHEGFP and hPTH-Cys were prepared to develop immunoassays for PTH detection in clinical samples. Initial experiments show promise for these analogs for use in CZELIF based immunoassays. The analogs present a number of distinctive advantages for clinical assays and can be used to develop several immunoassay platforms.

Introduction: Pulmonary Surfactant reduces surface tension in the terminal airways thus facilitating breathing and contributes to host's innate immunity. Surfactant Proteins (SP) A, B, C and D were recently identified as inherent proteins of the CNS. Aim of the study was to investigate cerebrospinal fluid (CSF) SP levels in hydrocephalus patients compared to normal subjects. Patients and methods: CSF SP A-D levels were quantified using commercially available ELISA kits in 126 patients (0±84 years, mean 39 years). 60 patients without CNS pathologies served as a control group. Hydrocephalus patients were separated in aqueductal stenosis (AQS, n = 24), acute hydrocephalus without aqueductal stenosis (acute HC w/o AQS, n = 16) and idiopathic normal pressure hydrocephalus (NPH, n = 20). Furthermore, six patients with pseudotumor cerebri were investigated. Results: SP AÐD are present under physiological conditions in human CSF. SP-A is elevated in diseases accompanied by ventricular enlargement (AQS, acute HC w/o AQS) in a significant manner (0.67, 1.21 vs 0.38 ng/ml in control, p<0.001). SP-C is also elevated in hydrocephalic conditions (AQS, acute HC w/o AQS; 0.87, 1.71 vs. 0.48 ng/ml in controls, p<0.001) and in Pseudotumor cerebri (1.26 vs. 0.48 ng/ml in controls, p<0.01). SP-B and SP-D did not show significant alterations. Conclusion: The present study confirms the presence of SPs in human CSF. There are significant changes of SP-A and SP-C levels in diseases affecting brain water circulation and elevation of intracranial pressure. Cause of the alterations, underlying regulatory mechanisms, as well as diagnostic and therapeutic consequences of cerebral SP's requires further thorough investigations.

Les immunoessais en phase solide sont utilisés pour le diagnostic in vitro afin de détecter ou de quantifier une molécule dans un échantillon biologique. Ils s'appuient sur l'interaction spécifique entre un antigène et un anticorps. Habituellement, des anticorps spécifiques aux antigènes à détecter sont immobilisés sur une surface solide pour capturer les antigènes d'intérêt et les séparer du reste de l'échantillon.Lors du développement d'un immunoessai, la sensibilité, la spécificité et le temps d’analyse sont optimisés par le choix - classiquement empirique - de ligands, de supports solides, de débits,… Une meilleure compréhension et prédiction des interactions moléculaires complexes se produisant au cours d’un immunoessai seraient utiles pour : identifier les paramètres critiques des immunoessais, simplifier et accélérer le processus d’identification des meilleures conditions opératoires et améliorer les immunoessais existants.L'instrument VIDAS®, commercialisé par bioMérieux, est l'un des systèmes d’immunoessais les plus utilisés dans les laboratoires cliniques. Dans ce travail de thèse, deux outils expérimentaux basés sur la chromatographie inverse sont construits et testés. Un modèle prédictif de la cinétique d'interaction anticorps/antigène est développé. Les outils expérimentaux, fonctionnant dans des conditions très proches du VIDAS®, sont utilisés pour valider le modèle et estimer ses paramètres caractérisant les interactions anticorps/antigène à partir de courbes expérimentales. Dans l’avenir et à partir des résultats, un des outils expérimentaux associé au modèle pourra être utilisé par bioMérieux pour concevoir des systèmes d’immunoessais / Solid-phase immunoassays are used for in vitro diagnostic to detect the presence or measure the concentration of a molecule of interest in a biological sample. They rely on the specific interaction between an antigen and an antibody. Usually, antibodies specific to the antigens to be detected are immobilized on a solid surface to capture the antigens of interest and separate them from the rest of the sample components. During solid-phase immunoassay development, sensitivity, specificity and time-to-result need to be optimized through the choice of dedicated ligands, solid supports, flow rates,… Classically, these choices are made empirically. A better understanding and prediction of the complex molecular interactions that occur in the different steps of a diagnostic immunoassay is likely to be useful to: identify the critical parameters of immunoassays, simplify and speed-up the process of identification of the best immunoassay conditions and improve the immunoassays currently available. The VIDAS® instrument, commercialized by bioMérieux is one of the most widely used immunoassay system in clinical laboratories worldwide. In this PhD work, two experimental tools based on inverse chromatography are built and tested. A predictive model of antibody/antigen interaction kinetics in immunoassays is developed. The experimental tools which mimic VIDAS® process conditions are used to validate the predictive model and to estimate model parameters characterizing antibody/antigen interaction kinetics from experimental curves. In the future, based on the results, one of the experimental tools associated with the model could be used by bioMérieux to design immunoassay systems

Until first half of the XX century, sickle cell disease was practically limited to the malaria endemic areas and countries having known an important surge of African slaves. Today, migratory flows and progress of medicine have modified considerably the distribution of sickle cell disease which is from now on a frequent affection in Western Europe. The preventive implementation of medical care makes it possible to reduce morbidity and mortality associated with this pathology. Stake of a medical policy and economic interests, neonatal screening for hemoglobin disorders justifies then fully the implementation of powerful and adapted means. In order to initiate a newborn screening programme in our centre, we developed various immunological tests allowing to identify the sickle hemoglobin. We first of all developed an indirect immunoassay and led a population study on 46082 Belgian newborns and 1825 neonates from Central Africa. The performances of this assay were improved thereafter by conceiving a competitive test. Next, for reasons independent of our will, we had unfortunately to abandon the immunological approach. This methodology was thus supplanted in our center by an innovative method for this indication: the mass spectrometry. Our promising results currently authorize us to perennialize our policy in the neonatal screening for sickle cell disease and open the way for new developments in other fields. / Jusquà la première moitié du XXe siècle, la drépanocytose se limitait pratiquement aux zones dendémie palustre et aux pays ayant connu un important afflux desclaves dorigine africaine. Aujourdhui, les flux migratoires et les progrès de la médecine ont considérablement modifié la distribution de cette maladie qui est désormais une affection fréquente en Europe occidentale. La prise en charge précoce permet de réduire la morbidité et la mortalité associées à cette maladie. Enjeu dune politique sanitaire et dintérêts économiques, le dépistage néonatal de la drépanocytose justifie donc ainsi pleinement la mise en uvre de moyens performants et adaptés. Afin dinitier un programme de dépistage au sein de notre centre, nous avons initialement développé divers tests immunologiques permettant didentifier lhémoglobine anormale. Nous avons tout dabord mis au point un immunoessai indirect et conduit une étude de population sur 46082 nouveau-nés belges et 1825 bébés originaires dAfrique centrale. Les performances de lessai ont par la suite été améliorées en concevant un test compétitif. Lapprovisionnement laborieux danticorps nécessaires aux tests de détection a par la suite entravé notre programme. En effet, la commercialisation en a été interrompue et la production danticorps monoclonaux par nos moyens propres na pas été couronnée du succès escompté. Lapproche immunologique du dépistage néonatal de la drépanocytose a ainsi été supplantée dans notre centre par une méthode novatrice pour cette indication : la spectrométrie de masse. Nos résultats prometteurs nous autorisent actuellement à pérenniser notre nouvelle façon de faire dans le dépistage néonatal de la drépanocytose et ouvre la voie pour de nouveaux développements dans dautres domaines.

A method for extraction of therapeutic proteins from dried blood spots (DBS) followed by quantification on Gyrolab(TM) has been developed. The method makes it possible to measure the concentration of the analyte in the range 100-6000 ng/mL. The procedure can generate full analytical information from 15 μL blood originally sampled from a subject. The modest sample requirements allows for sampling a full pre-clinical pharmacokinetic profile from a single mouse. This may allow for reduced usage of animals during preclinical development of new therapeutic proteins in accordance with the 3R’s, replace, refine and reduce.

This thesis describes the synthesis and characterization of metal-encoded polystyrene microspheres with a narrow size distribution designed for mass cytometry-based immuno- and oligonucleotide-assays. These particles were prepared by multiple stage dispersion polymerization techniques using polyvinylpyrrolidone (PVP) as a steric stabilizer. As a cytometeric technique, mass cytometry necessitated metal-encoded microspheres to perform the same roles of fluorescent microspheres used in conventional flow cytometry. The first role of the microsphere was to be able to act as a platform (classifier microspheres) for bioassays. Secondly, the microspheres should be suitable for mass cytometry machine calibration as standards. To perform these roles, metal-encoded microspheres were required to have certain size, functionality and metal content criteria. Lanthanide elements were chosen as the metals for encoding the microspheres for their low natural abundance in biological systems and for their similar chemistry. My goal was to employ two-stage dispersion polymerization, of styrene in ethanol, to introduce the lanthanide salts along with excess acrylic acid in the second stage, one hour after the initiation. Acrylic acid deemed to serve as a ligand for the lanthanide ions, through its carbonyl group, so the lanthanide ions get incorporated into the microsphere while acrylic acid is copolymerizing with styrene. Using two-stage dispersion polymerization, I could synthesize lanthanide encoded microspheres with narrow size distribution and high lanthanide content. However the lanthanide content distributions were unexpectedly much broader than the size distribution obtained. In addition, I could not attach biomolecules to the surface of such particles. In an attempt to improve the characteristics of these microspheres, I employed modified versions of multiple stage dispersion polymerization and seeded emulsion polymerization to grow functional polymer shell on the surface of the particles prepared by dispersion polymerization. Moreover, I coated the lanthanide encoded microspheres with silica shell which enabled me to grow another layer of functional-silica. Consequently, I could use these particles as classifier microspheres for mass cytometry-based immunoassays as well as fluorescence-based oligonucleotide-assays.

This thesis describes the synthesis and characterization of metal-encoded polystyrene microspheres with a narrow size distribution designed for mass cytometry-based immuno- and oligonucleotide-assays. These particles were prepared by multiple stage dispersion polymerization techniques using polyvinylpyrrolidone (PVP) as a steric stabilizer. As a cytometeric technique, mass cytometry necessitated metal-encoded microspheres to perform the same roles of fluorescent microspheres used in conventional flow cytometry. The first role of the microsphere was to be able to act as a platform (classifier microspheres) for bioassays. Secondly, the microspheres should be suitable for mass cytometry machine calibration as standards. To perform these roles, metal-encoded microspheres were required to have certain size, functionality and metal content criteria. Lanthanide elements were chosen as the metals for encoding the microspheres for their low natural abundance in biological systems and for their similar chemistry. My goal was to employ two-stage dispersion polymerization, of styrene in ethanol, to introduce the lanthanide salts along with excess acrylic acid in the second stage, one hour after the initiation. Acrylic acid deemed to serve as a ligand for the lanthanide ions, through its carbonyl group, so the lanthanide ions get incorporated into the microsphere while acrylic acid is copolymerizing with styrene. Using two-stage dispersion polymerization, I could synthesize lanthanide encoded microspheres with narrow size distribution and high lanthanide content. However the lanthanide content distributions were unexpectedly much broader than the size distribution obtained. In addition, I could not attach biomolecules to the surface of such particles. In an attempt to improve the characteristics of these microspheres, I employed modified versions of multiple stage dispersion polymerization and seeded emulsion polymerization to grow functional polymer shell on the surface of the particles prepared by dispersion polymerization. Moreover, I coated the lanthanide encoded microspheres with silica shell which enabled me to grow another layer of functional-silica. Consequently, I could use these particles as classifier microspheres for mass cytometry-based immunoassays as well as fluorescence-based oligonucleotide-assays.

Submitted by JULIANA MARIA QUINALHA DE SOUZA null (jumquinalha@hotmail.com) on 2016-09-30T18:17:53Z No. of bitstreams: 1 Dissertação_mestrado_Juliana_Quinalha.pdf: 2370086 bytes, checksum: 98ac8515c64de9b4f7df6656025e2d3d (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-10-04T16:46:31Z (GMT) No. of bitstreams: 1 souza_jmq_me_bot.pdf: 2370086 bytes, checksum: 98ac8515c64de9b4f7df6656025e2d3d (MD5) / Made available in DSpace on 2016-10-04T16:46:31Z (GMT). No. of bitstreams: 1 souza_jmq_me_bot.pdf: 2370086 bytes, checksum: 98ac8515c64de9b4f7df6656025e2d3d (MD5) Previous issue date: 2016-08-29 / Introdução: A Doença Trofoblástica Gestacional (DTG) compreende dois tipos clínicos: mola hidatiforme (MH) e neoplasias trofoblásticas gestacionais (NTG). A dosagem do hCG é o parâmetro mais importante para detecção da persistência de trofloblasto ativo na parede do útero ou em outros locais do organismo. Objetivo: Comparar concentrações séricas do hormônio hCG em pacientes com DTG utilizando duas variações do método de quimioluminescência comercialmente disponíveis em nosso centro. Métodos: Este estudo incluiu pacientes com DTG avaliadas e acompanhadas no Centro de Doenças Trofoblásticas da Faculdade de Medicina de Botucatu (CDTB) – UNESP, de novembro de 2014 a outubro de 2015. Amostras de soro das pacientes foram testadas para dosagem de hCG em duas variações do método de quimioluminescência ARCHITECT® i2000 SR e IMMULITE® 2000 Xpi. Concentrações séricas de hCG foram comparadas contra a hipótese nula e a concordância clínica foi determinada em dois momentos: admissão da paciente e evolução pela curva de hCG de acordo com os valores de hCG entre os dois equipamentos. Resultados: 73 pacientes com DTG foram incluídas no estudo. Destas, 45 tinham MH e remissão espontânea, enquanto 28 tiveram NTG. Uma boa concordância nos valores médios de hCG entre IMMULITE® 2000 Xpi e ARCHITECT® i2000 SR quando hCG <100 mUI/mL. Para valores de hCG >100 mUI/mL, houve diferença significativa entre ensaios (p<0,05), com valores medidos pelo ARCHITECT® i2000 SR sendo maior que aqueles medidos pelo IMMULITE® 2000 Xpi em pacientes com MH e remissão espontânea ( Immulite = 0.79Architect ; p < 0,01 ) ; R2 = 90 % ) e NTG ( Immulite = 0,51Architect ; p < 0,01 ) ; R2 = 98 % ). As condutas clínicas no momento da admissão das pacientes foram concordantes em 100% dos casos [73/73(100%); kappa1; p<0,001] e pela evolução da curva de hCG, a concordância observada foi de 98% [71/72(98%); kappa 0,93; p<0,001].Conclusão: O uso do equipamento IMMULITE® é recomendado para monitoramento de pacientes com DTG, entretanto, ARCHITECT® apresentou desempenho analítico e coerência clínica em relação aos resultados de hCG, o que faz deste equipamento uma boa alternativa. / Introduction: Gestational trophoblastic disease (GTD) is a group of disorders spanning the conditions that range from hydatidiform mole (HM) to gestational trophoblastic neoplasia (GTN). Determining human chorionic gonadotrophin (hCG) serum levels is crucial for the early detection and optimal management of GTN. Objective: To compare serum hCG levels in patients with GTD using 2 commercially available hCG immunoassays. Methods: Serum samples were obtained from patients with GTD attending the Botucatu Trophoblastic Diseases (Botucatu Medical School, São Paulo State University- Unesp) from November 2014 to October 2015. Serum hCG levels were measured with both IMMULITE® 2000 XPi and ARCHITECT® i2000 chemiluminescence tests. Serum hCG levels were compared against the null hypothesis. Agreement with clinical management was determined by comparing baseline hCG measures as well as the hCG curves obtained with both assays. Results: Seventy-three patients with GTD were included in the analysis. Of these, 45 had HM and experienced spontaneous remission, while 28 had GTN. There was a perfect (zero difference) agreement in mean hCG levels between IMMULITE® 2000 XPi and ARCHITECT® i2000 when hCG < 100 mUI/mL. For hCG values greater than 100 mUI/mL, there was a significant difference between assays (p < 0.05), with levels measured via ARCHITECT® i2000 being higher than those measured with IMMULITE® 2000 XPi in patients with spontaneous remission (Immulite = 0.79Architect; p < 0.01); R2 = 90%) and GTN (Immulite = 0,51Architect; p < 0,01); R2 = 98%). Baseline test results agreed in all cases (73/73(100%); kappa1;p<0,001] and hCG curve agreement was 98% [71/72(98%); kappa0,93;p<0,001].Conclusion: IMMULITE® has been the assay recommended for diagnosing and monitoring patients with GTD. However, our results suggest that as ARCHITECT® and IMMULITE® show similar performance in measuring hCG levels and determining clinical management, ARCHITECT® may be used an alternative.

Orientador: Izildinha Maestá / Resumo: Introdução: A Doença Trofoblástica Gestacional (DTG) compreende dois tipos clínicos: mola hidatiforme (MH) e neoplasias trofoblásticas gestacionais (NTG). A dosagem do hCG é o parâmetro mais importante para detecção da persistência de trofloblasto ativo na parede do útero ou em outros locais do organismo. Objetivo: Comparar concentrações séricas do hormônio hCG em pacientes com DTG utilizando duas variações do método de quimioluminescência comercialmente disponíveis em nosso centro. Métodos: Este estudo incluiu pacientes com DTG avaliadas e acompanhadas no Centro de Doenças Trofoblásticas da Faculdade de Medicina de Botucatu (CDTB) - UNESP, de novembro de 2014 a outubro de 2015. Amostras de soro das pacientes foram testadas para dosagem de hCG em duas variações do método de quimioluminescência ARCHITECT® i2000 SR e IMMULITE® 2000 Xpi. Concentrações séricas de hCG foram comparadas contra a hipótese nula e a concordância clínica foi determinada em dois momentos: admissão da paciente e evolução pela curva de hCG de acordo com os valores de hCG entre os dois equipamentos. Resultados: 73 pacientes com DTG foram incluídas no estudo. Destas, 45 tinham MH e remissão espontânea, enquanto 28 tiveram NTG. Uma boa concordância nos valores médios de hCG entre IMMULITE® 2000 Xpi e ARCHITECT® i2000 SR quando hCG <100 mUI/mL. Para valores de hCG >100 mUI/mL, houve diferença significativa entre ensaios (p<0,05), com valores medidos pelo ARCHITECT® i2000 SR sendo maior que aqueles medidos pelo... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre