Les muqueuses représentent la principale voie de contamination par le virus de l'immunodéficience humaine 1 (VIH-1) indiquant qu'une protection au niveau des muqueuses serait nécessaire dans la prévention et le développement d'un vaccin efficace. La prise en charge du VIH-1 par l'intermédiaire de la gp120 se fait, grâce à des lectines de type C, au niveau de la muqueuse par les cellules de Langerhans (Langerine) et au niveau de la sous-muqueuse par les cellules dendritiques (DC-SIGN). Notre but était d'analyser la présence d'anticorps capables de bloquer l'interaction entre le virus et les lectines. Nous avons développé des tests spécifiques pour l'étude de la liaison de la gp120 du VIH-1 avec la Langerine et le DC-SIGN. Grâce à ces tests, nous avons pu caractériser des anticorps, dirigés contre la Langerine ou le DC-SIGN, permettant le blocage de la fixation de la gp120 sous forme conformationnelle. Nous avons utilisé ces tests validés pour étudier la fréquence d’anticorps bloquants dans le sérum de 110 individus séropositifs et 30 individus séronégatifs. Seulement peu de sujets infectés ont développé ce genre d'anticorps durant l'infection par le VIH-1. Nous avons pour la première fois décrit des anticorps bloquants chez des sujets séropositifs avec la capacité potentielle d’interférer dans la transmission des particules virales aux cellules dendritiques. Ces résultats permettent d'espérer la conception de nouvelles stratégies pour bloquer la transmission du VIH-1 au niveau des muqueuses et prévenir la dissémination du virus. Ces tests permettront de doser l'inhibition de la liaison de la gp120 soit à la Langerine soit au DC-SIGN et d'étudier différentes cohortes de sujets séropositifs et séronégatifs mais fortement exposés (HEPS) / Human immunodeficiency virus-1 (HIV-1) is primarily transmitted sexually through mucosal surfaces, indicating that protection at mucosal sites may be crucial in prevention and in the development of an effective conventional vaccine against HIV-1. The HIV-1 gp120 is recognized by the C-type lectins of Langerhans cells (LCs), Langerin, in the mucosa and by the C-type lectins of dendritic cells (DCs), DC-SIGN in the sub-mucosa. Our aim was to analyze the presence of antibody able to block HIV / Lectins interactions. We have developed specific assays for the study of the binding of HIV-1 gp120 on human Langerin and DC-SIGN. With these assays, we have been able to characterize specific DC-SlGN and Langerin antibodies able to block fixation of conformational gp120 envelope protein. We have used our validated assays to monitor the prevalence of blocking antibodies in the sera of one hundred and ten HIV-1-infected individuals and thirty HIV-1 non-infected individuals. Only few patients have developed such type of antibodies during their infection. This is the first report which describes blocking antibodies in patients with the potential capacity to interfere during the mucosal transmission of HIV particles to mucosal DCs. The results provide a framework for the design of effective strategies to block local transmission and prevent HIV-1 spread. These tests will allow the measuring of the inhibition of gp120 binding to langerin or to DC-SIGN and the screening of different cohorts of HIV-1 positive subjects and HIV-1 negative but highly exposed subjects (HEPS)

Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-03-08T19:19:08Z No. of bitstreams: 1 117110029.pdf: 698994 bytes, checksum: ceaed51c4f43c77fa5dcfffa7ec011b5 (MD5) / Made available in DSpace on 2015-03-08T19:19:08Z (GMT). No. of bitstreams: 1 117110029.pdf: 698994 bytes, checksum: ceaed51c4f43c77fa5dcfffa7ec011b5 (MD5) Previous issue date: 2013-08-09 / Os vírus são importantes agentes patogênicos de várias espécies animais, entre elas bovinos. No Brasil, diversos agentes virais foram descritos causando infecções no rebanho bovino, e produzindo perdas econômicas significativas. A identificação dos animais infectados por um vírus pode ser realizada de diferentes formas; no entanto, a confirmação definitiva requer a demonstração do agente ou da resposta imune. Para isto, vários métodos com capacidade de detectar a partícula viral, atividade biológica, genoma, antígenos virais, ou então a resposta imune específica foram desenvolvidos. Os imunoensaios são testes amplamente utilizados na rotina laboratorial para detecção de antígenos virais em amostras clínicas ou de pesquisa. Estes ensaios apresentam boa sensibilidade, especificidade e facilidade de execução. A metodologia dos imunoensaios tem como base, o emprego de anticorpos monoclonais ou policlonais específicos para os antígenos virais. Assim sendo, o objetivo do presente estudo foi produzir anticorpos policlonais para alguns vírus bovinos, e avaliar a reatividade destes em testes de imunofluorescência, imunoperoxidase e slot blot. Para isto, cepas e/ou isolados do herpesvírus bovino tipo 1 (BoHV-1), herpesvírus bovino tipo 2 (BoHV-2), herpesvírus bovino tipo 5 (BoHV-5), herpesvírus bovino tipo 5 gE deletado (BoHV-5 gEΔ), vírus da diarreia viral bovina (BVDV), vírus respiratório sincicial bovino (BRSV), vírus da língua azul (BTV) e vírus da vaccínia (VACV) foram amplificados em cultivo celular e o sobrenadante utilizado para imunizar coelhos. Os animais foram imunizados cinco vezes pela via subcutânea, e cinco dias após o último reforço coletou-se sangue. O soro foi separado do sangue por centrifugação. O soro foi diluído em PBS (1:100 a 1:204.800) e utilizado como anticorpo primário nos ensaios de imunofluorescência, imunoperoxidase e slot blot. A diluição de trabalho foi selecionada pela diluição que produziu reação específica nas células infectadas, e sinal fraco ou ausente nas células controle. Os antissoros apresentaram maior reatividade na técnica de imunoperoxidase do que na imunofluorescência e slot blot. Ainda, para os antissoros do BoHV-1, BoHV-5, BVDV e BRSV demonstrou-se a reatividade com amostras heterólogas nos ensaios de imunofluorescência e imunoperoxidase. Conclui-se que os anticorpos policlonais produzidos em coelhos possuem elevadas concentrações de anticorpos específicos, o que foi detectado pela reatividade nos ensaios de imunofluorescência, imunoperoxidase e slot blot. Desta maneira, estes reagentes podem ser considerados uma importante ferramenta para a detecção e caracterização de vários vírus bovinos na rotina de diagnóstico e pesquisa. / The viruses are significant important pathogenic agents of several animal species, including cattle. In Brazil, several viral agents causing infections have been described in cattle and they produce significant economic losses. The identification of animals infected by a virus can be performed in different ways; however, definitive confirmation requires demonstration of the agent or immune response. For this purpose, various methods with the capacity to detect the viral particle, biological activity, genome, viral antigens, or specific immune response have been developed. Immunoassays are widely used in laboratory routine for detection of viral antigens in clinical or research. These assays exhibit good sensitivity, specificity and easy for implantation. The immunoassay methodologies are based on the employment of monoclonal or polyclonal antibodies specific to the viral antigens. Therefore, the aim of this study was to produce polyclonal antibodies for some bovine virus, and evaluate their reactivity in immunofluorescence, immunoperoxidase and slot blot tests. For this purpose, strains and/or isolates of bovine herpesvirus type 1 (BoHV-1), bovine herpesvirus type 2 (BoHV-2), bovine herpesvirus type 5 (BoHV-5), bovine herpesvirus type 5 gE deleted (BoHV-5 gEΔ), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bluetongue virus (BTV), and vaccinia virus (VACV) were amplified in cell culture and the supernatant were used to immunize rabbits. The animals were immunized five times by the subcutaneous route, and five days after the last boost the blood was collected. The serum was obtained by centrifugation. The serum was diluted (1:100 a 1:204.800) and used as primary antibodies in the immunofluorescence, immunoperoxidase and slot blot assays. The working dilution was selected among those produced specific reaction with infected cells and absent or weak background in control cells. The antiserum showed higher reactivity in immunoperoxidase technique than the immunofluorescence and slot blot. The antiserum of the BoHV-1, BoHV-5, BVDV and BRSV presented the reactivity when tested with eterologous isolates in immunofluorescence, immunoperoxidase assays. In summary, that the polyclonal antibodies raised in rabbits have high concentrations of specific antibodies, which were demonstrated by the reactivity in immunofluorescence, immunoperoxidase and slot blot assays. Additionally, these reagents can be considered an important tool for the detection and characterization of various bovine viruses in diagnostic and research routine.

This thesis presents a light scattering-based method for biomarker detection, which could potentially be used for the quantification of multiple biomarkers specific to B-cell malignancies. This method uses fabricated gold nanoparticle probes to amplify inelastic light scattering in a process referred to as surface-enhanced Raman scattering. These gold nanoparticle probes were conjugated to antibodies for specific and targeted molecular binding. The spectrum of the amplified inelastic light scattering was detected using a spectrometer and a detector. To detect the light scattering signal from the gold nanoparticle probes, several commercial Raman spectrometer instruments were evaluated. Initial results from these evaluations are presented in this thesis. After system evaluation, a custom Raman microscope system was designed, built, and tested. This system was used for the development of a surface-enhanced Raman spectroscopy-based immunoassay. The development of this assay confirms the successful design of gold nanoparticle probes for the specific targeting and detection of immunoglobulins. The immunoassay also shows promise for the simultaneous detection of multiple biomarkers specific to B-cell malignancies.

The objective of this dissertation is to improve the performance of surface acoustic wave (SAW) biosensors for use in point-of-care-testing (POCT) applications. SAW biosensors have the ability to perform fast, accurate detection of an analyte in real time without the use of labels. However, the technology suffers from the inability to differentiate between specific and non-specific binding. Due to this limitation, direct testing of bodily fluids using SAW sensors to accurately determine an analyte's concentration is difficult. In addition, these sensors are challenged by the need to detect small concentrations of a biomarker that are typically required to give a clinical diagnosis. Sensitivity, selectivity and reliability are three critical aspects for any sensing platform. To improve sensitivity the delay path of a SAW sensor has been modified with microcavities filled with various materials. These filled cavities increased sensitivity by confining wave energy to the surface by way of constructive interference and waveguiding. Thus, the improved sensitivity will result in a lower limit of detection. In addition, insertion loss is decreased as a consequence of increased wave confinement to the surface. Sensor selectivity and reliability are adversely affected by non-specific binding of unwanted species present in a sample. To address this issue a multifunctional SAW sensor is presented. The sensor consists of two SAW delay lines oriented orthogonal to each on ST-quartz in order to generate two distinct wave modes. One wave mode is used for sensing while the other is used to remove loosely bound material. By using the same transduction mechanism for both removal and sensing, the sensor chip is simplified and complex electronics are avoided. The findings of this research involve the technological advances for SAW biosensors that make their use in POCT possible.

The goal of this research is to investigate computational and experimental techniques to effectively analyze microscale fluid dynamics, transport, and mixing of an analyte-antibody system. This work is applicable to the development of an in-plane, passive mixer component of a miniature antibody-based sensor suitable for environmental monitoring, food testing, and medical diagnostics. The computational methods allow the efficient evaluation of microchannel designs to enhance analyte-antibody binding, which may reduce the time and cost required for experimental trials. We describe a computational algorithm to solve the governing equations for microscale fluid flow and transport in complex 2-D domains created through a graphical user interface. We implement the particle strength exchange method to solve the convection-diffusion-reaction equations, coupled to the boundary element method to compute the velocity field from the steady state Stokes equations. We validate the numerical methods by comparison to analytical and finite element method solutions. Because the chosen methods require no internal mesh, our algorithm provides an efficient alternative to grid-based methods when solving transport in complex geometries with internal obstacles. We characterize two fluorescein-antibody clones through competitive ELISA experiments and demonstrate the quenching effect of the antibodies with a fluorescence spectrophotometer. We describe a microchannel flow system to image the quenching of fluorescence by the antibody when fluorescein and fluorescein-antibody solutions are injected into separate inlets of the microchannel. We correlate the fluorescence intensity of microscope images of fluorescein flowing through the microchannel to concentrations of fluorescein to establish a calibration curve. This system provides a method to visualize and quantitatively analyze the mixing and reaction in a microfluidic device. We test the numerical methods by comparing the experimentally determined fluorescein concentration to the outlet amount numerically predicted by the computational model under identical conditions and find good agreement between the two fluorescein concentration profiles. We complete the transport-reaction computation in a set of microchannels with cylindrical obstructions. We find that decreasing the channel width and increasing the fluid path length by placing the obstruction on the walls is more effective than placing free-standing obstructions within the channel to enhance the fluorescein and fluorescein-antibody reaction. / acase@tulane.edu

<p><strong>Objective</strong><em>:</em> Estimation of glomerular filtration rate (GFR) is an important tool in the diagnosis and management of chronic kidney disease. Today creatinine is the most frequently used marker for kidney function though several studies indicate that cystatin C is a superior marker. The purpose of this study was to validate Abbott Diagnostics turbidimetric cystatin C assay and enzymatic creatinine assay.</p><p><strong>Methods</strong><em>:</em> The validation was performed by studies of CV for the two methods and correlations between the two and other available methods for assessing GFR. The stability of cystatin C at room temperature was also evaluated.</p><p><strong>Results</strong><em>: </em>Both methods showed good precision. The Abbott cystatin C assay generally gave lower values and thereby higher estimated GFRs than the correlated Gentian method. The Abbott enzymatic creatinine assay gave higher values than the correlated Jaffe method. Those results are generally unexpected, but in this study the cause is an automatically applied negative intercept used together with the Jaffe method. Cystatin C showed high stability when stored at room temperature.</p><p><strong>Conclusions</strong><em>:</em> Estimated GFRs tend to differ depending on the choice of method for analyzing cystatin C or creatinine and this study gives an overview of the range of variation. The study also enlightens the need for an international calibrator for the cystatin C methods presented by different manufacturers.</p>

<p>Nuts of all kinds are common ingredients in food. For nut allergy sufferers the frequent use of nuts cause problems and "hidden" nuts in food products may elicit allergic reaction when such foods are consumed. Methods for detecting and quantifying walnut (and other nuts) with high sensitivity and specificity are therefore very important.</p><p>The objective of this project was to verify the specificity of a rabbit antiserum against walnut with immunodiffusion and to determine the size of the dominant walnut antigens with Western blotting. In addition, a commercial sandwich ELISA for walnut quantification was validated and compared with a qualitative real-time PCR.</p><p>The rabbit antiserum proved to be less specific but after absorption with cross-reacting nuts and seeds it showed high specificity. The ELISA kit reacted, except for walnut, with pecan and slightly with other nuts and seeds tested. The PCR showed an absolute specificity to walnut. As low levels as 2.5mg walnut/kg can be quantified with the ELISA. This is 8 to 100 fold less than with the PCR method. It is therefore concluded that the ELISA kit is more sensitive than the PCR method but the PCR method is more specific than the ELISA kit.</p>

Objective: Estimation of glomerular filtration rate (GFR) is an important tool in the diagnosis and management of chronic kidney disease. Today creatinine is the most frequently used marker for kidney function though several studies indicate that cystatin C is a superior marker. The purpose of this study was to validate Abbott Diagnostics turbidimetric cystatin C assay and enzymatic creatinine assay. Methods: The validation was performed by studies of CV for the two methods and correlations between the two and other available methods for assessing GFR. The stability of cystatin C at room temperature was also evaluated. Results: Both methods showed good precision. The Abbott cystatin C assay generally gave lower values and thereby higher estimated GFRs than the correlated Gentian method. The Abbott enzymatic creatinine assay gave higher values than the correlated Jaffe method. Those results are generally unexpected, but in this study the cause is an automatically applied negative intercept used together with the Jaffe method. Cystatin C showed high stability when stored at room temperature. Conclusions: Estimated GFRs tend to differ depending on the choice of method for analyzing cystatin C or creatinine and this study gives an overview of the range of variation. The study also enlightens the need for an international calibrator for the cystatin C methods presented by different manufacturers.

Nuts of all kinds are common ingredients in food. For nut allergy sufferers the frequent use of nuts cause problems and "hidden" nuts in food products may elicit allergic reaction when such foods are consumed. Methods for detecting and quantifying walnut (and other nuts) with high sensitivity and specificity are therefore very important. The objective of this project was to verify the specificity of a rabbit antiserum against walnut with immunodiffusion and to determine the size of the dominant walnut antigens with Western blotting. In addition, a commercial sandwich ELISA for walnut quantification was validated and compared with a qualitative real-time PCR. The rabbit antiserum proved to be less specific but after absorption with cross-reacting nuts and seeds it showed high specificity. The ELISA kit reacted, except for walnut, with pecan and slightly with other nuts and seeds tested. The PCR showed an absolute specificity to walnut. As low levels as 2.5mg walnut/kg can be quantified with the ELISA. This is 8 to 100 fold less than with the PCR method. It is therefore concluded that the ELISA kit is more sensitive than the PCR method but the PCR method is more specific than the ELISA kit.

The purpose of this investigation was to create and optimise a capture assay for the detectionof anti-drug antibodies (ADA) in human plasma, using Biacore. We also dealt with the nonspecificplasma binding to mouse-derived anti-biotin which may occur in the capture assay.By paying attention to these things we aimed at reaching as high sensitivity as possible for theADA detection. The capture assay also benefited and gained flexibility from using the same regenerationsolution irrespective of drug and from having a composition that minimises the risk ofdamaging drug epitopes.