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Implementation of a Chemiluminescent Immunoassay for S. Aureus Protein A in a Cd Microfluidic Device

The object of this thesis, carried out at the Centre de Recherche en Infectiologie in Quebec City (Canada) directed by Prof. M. Bergeron, has been the development of a sandwich ELISA immunoassay method for the determination of protein A of Staphylococcus aureus with revelation with detection by imaging chemiluminescente.S. aureus is a bacterium subject of intense study since it is a major cause of nosocomial infections, so there is certainly a need to develop methods and diagnostic tools for the rapid detection of infection by S. aureus. In this work we assessed the possibility of determining by a staphylococcal protein A immunoassay method. It therefore represents a suitable target for a diagnostic method for the detection of infections by S. aureus and its proteinaceous nature makes it particularly suited to the determination by an immunological method ELISA noncompetitive heterogeneous. In these methods the protein is bound with a specific capture antibody immobilized on a suitable support (es.una microtiter plate) and detected by a second antibody (detection antibody) labeled with an enzyme. In this thesis have been used detection antibodies labeled with the enzyme horseradish peroxidase, detected by spectrophotometric or chemiluminescence after the addition of suitable substrates enzimatici.Un important aspect of this thesis is the implementation of the immunometric method with detection by imaging of chemiluminescence in a microfluidic system on CD (represented in the figure) previously developed at the Centre de Recherche en Infectiologie, which uses centrifugal force to produce the flow of sample and reagents needed for the various stages of the analysis, and in that He had previously been used only for analysis based on hybridization of nucleic acids with detection by imaging fluorescenza.Terminata analysis, the slide is disconnected from the microfluidic and inserted into the instrument measures for imaging (fluorescence or chemiluminescence). To power to perform the measurement of the antibody detection by imaging chemiluminescence after the addition of a suitable enzyme substrate (for peroxidase, a system luminol / oxidant / enhancer) was also constructed a system constituted by an optical microscope coupled to a chamber thermoelectrically cooled CCD. For quantitative analysis, the images thus obtained were processed using an analysis software image. During the thesis work was first carried out a preliminary optimization of the experimental conditions (eg concentrations of reactants, saturating agent concentration, incubation times) for the immunometric determination of protein A using a format analytical conventional (microtiter plate) and using spectrophotometric detection techniques and then chemiluminescent. To transfer the analysis in microfluidic system of CD were then evaluated the optimal conditions for immobilization of capture antibody on the support used in this assay system (instead of glass as in polystyrene microtiter plates). To obtain the immobilization of the antibody through the formation of covalent bonds have been used commercial glass functionalized with aldehyde groups and the evaluation of the quality of immobilization was carried out by immobilizing antibodies labeled with fluorescein, visualized by means of fluorescence imaging using a scanner for microarray . The spots of antibody on the slide were obtained either manually or using an automated dispensing system for the production of microarrays, which enabled to obtain spot regular and highly riproducibili.L 'immunometric analysis of protein A was then performed using the system microfluidics on CD after a further optimization of the experimental conditions, made necessary by the different volumes of reagents used in this particular format. In the experimental conditions it was possible to achieve optimized reached a limit of detection (LOD) comparable with that obtained in microtiter plate, equal to 10 ng / ml is protein A. The value of detection limit suggests the potential applicability of this method to analysis diagnostiche.L 'use of chemiluminescent detection allows then to achieve low detection limits and to carry out the measurement by means of a relatively simple instrumentation. The results are therefore promising for a future use of this device in diagnostics for the analysis of multiple samples in a short time and at low cost.

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1 Chapter 1 INTRODUCTION 1.1 STAPHYLOCOCCUS AUREUS Staphylococci are responsible of many infections in humans (Fig.1.1). Figure 1.1 – main infections due to Staphylococci In particular S. aureus causes osteomyelitis, septic arthritis, endocarditis and serious skin infections. In association with S. epidermidis, it causes nosocomial acquired infections of surgical wounds and medical devices, while S. saprophiticus causes urinary tract infections. S. aureus is also responsible of food poisoning and toxic shock syndrome (Donald 1993). Staphylococci are Gram-positive cocci with 1 µm of diameter, which create clumps and grow in clusters pairs, less frequently in short chains. Staphylococci are, differently from

Laurea liv.II (specialistica)

Facoltà: Farmacia

Autore: Deborah Sebastianelli Contatta »

Composta da 53 pagine.


Questa tesi ha raggiunto 25 click dal 29/10/2012.

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