Thèse de doctorat : Parasitologie : Paris 12 : 2005. / Thèse uniquement consultable au sein de l'Université Paris 12 (Intranet). Titre provenant de l'écran-titre. Bibliogr. : 192 réf.

Anoplocephala perfoliata is a species of parasitic worm that belongs to a group known as cestodes, which specifically target equine animals. As with all types of tapeworms, these parasites infect the gastrointestinal tract of their host, with devastating and potentially fatal consequences. The current lack of a sensitive and specific test for this parasite means that it continues to go undetected, hense this project aims to develop a novel and rapid diagnostic test with high sensitivity and specificity to help increase detection, thus precluding economic loss in the equine industry. The project details the development of three unique detection platforms; an ELISA, a lateral flow assay and an impedimetric immunosensor, aimed to detect IgA in saliva, since IgA is the dominant immunoglobulin of the mucosal immune system. IgA was therefore believed to be the ideal marker for rapid, specific and early indication of infection with A. perfoliata. Diagnosis using saliva samples was an integral part of this project, since it would allow for non-invasive sampling, by non-skilled personnel. A highly sensitive ELISA-based detection system was developed in this project for the detection of 3 different types of IgA. The first ELISA was developed to detect non-specific or ‘total’ IgA levels. Using a sandwich ELISA format, IgA was detectable with a LoD of ~0.04 ng/ml. A second ELISA was developed using the crude excretory/secretory (E/S) antigen, cultured from A. perfoliata worms, which were obtained by a vet during post-mortem examination of infected horses. The crude antigen mix was then used to fabricate an ELISA to detect specific IgA in saliva, produced against the E/S antigens. The crude antigen was then employed in a series of SDS PAGE and western blot experiments, which revealed the 12/13 kDa antigen as the main antigen detected by IgA in saliva. The 12/13 kDa was then electroeluted and used to immunise rabbits, in order to obtain anti-12/13 kDa antibodies, which were later used to purify large quantities of the 12/13 kDa antigen from the crude antigen mix. This allowed for the fabrication of the third and final ELISA, to detect IgA specific to the 12/13 kDa antigen. The 3 ELISAs were optimised throughout this project to ensure the most ideal conditions, such as antibody concentrations, sample dilutions, sample diluents, incubation temperatures and times were employed to obtain maximum assay sensitivity, specificity and productivity in a commercial setting. Testing samples (n = 24) using all 3 ELISAs and then standardising the specific IgA levels against the non-specific IgA, allowed for a novel and reliable detection method for A. perfoliata to be developed. This diagnostic test was developed in partnership with Austin Davis Biologics Ltd., who in April 2014 launched a screening programme which now offers horse owners an accurate means of testing their horses for A. perfoliata infections accurately. The second detection platform developed during this project was a lateral flow assay, whereby an immunochromographic strip was used to measure IgA levels in saliva. The studies performed determined the optimal conditions as using 40 µl of a 1:1,000 dilution of saliva using PBS(T) 1% as the sample diluent. The capture and control antibody were used at a concentration of 0.2 mg/ml, which were coated on the nitrocellulose membrane using an automated dispensing system (BioDot). The conjugate was labelled using gold nanoparticles, since it does not require any substrates or wash steps and its aggregation allows for immediate visual detection. A LoD of ~47 ng/ml was obtained for this assay. The final detection system investigated as part of this project was a label-less impedimetric immunosensor, whereby IgA was detected by means of electrochemical impedance spectroscopy (EIS). Polyaniline was the conductive polymer chosen to coat the surface of the screen printed carbon electrode, since the amine groups could be utilised to immobilise biotin molecules. A biotin-avidin complex was employed to ensure the uniform immobilisation of the capture antibody. Using the capture and control antibody at a concentration of 50 µg/ml and 10 mM ferri-ferrocyanide as the redox solution, IgA concentrations over a range of 100 – 0 ng/ml were investigated by Electrochemical Impedance Spectroscopy (EIS).

No description available.

Coeliac disease is a chronic enteropathy triggered by gluten. Patients produce antibodies to gliadin and the autoantigen tissue transglutaminase (tTG). These anti-tTG autoantibodies are disease specific and used in diagnosis. The autoantibodies can be detected by immunofluorescence (the endomysial antibody tests) or by ELISA using recombinant tTG. In vivo tTG associates with fibronectin, which may account for the greater sensitivity of the endomysial antibody assay compared to the ELISA. This project had two aims:  to determine whether GST-tagged tTG bound fibronectin and then, using the fibronectin bound tTG, whether a two-tiered ELISA increased anti-tTG binding in coeliac disease patients. First fibronectin was coupled to a solid support and then incubated with tTG. This was then analysed using SDS-PAGE. Secondly, an ELISA with a two-tiered antigen coating was created by coupling tTG to Fn. This mimics the in vivo orientation of the antigen and could theoretically increase anti-tTG binding. Comparative ELISAs were then run to see if anti-tTG binding differed between tTG and fibronectin-coupled tTG antigen coatings. Results showed GST-tagged tTG bound fibronectin. Coupling of tTG to fibronectin gave no improved binding of anti-tTG. On the contrary, most patients tested had decreased anti-tTG binding compared to the normal tTG based ELISA.

No description available.

No description available.

ABSTRACT Background Calprotectin is a protein found in the cytoplasm of neutrophile granulocytes. In the course of inflammatory bowel disease (IBD), calprotectin is released during chronic inflammation in the gut. Activation of neutrophils during the inflammation is followed by activation and secretion of pro-inflammatory molecules such as calprotectin. Calprotectin is stable in stool up to 7 days and can therefore be used as a non-invasive marker for diagnosis, treatment and measurement of the disease activity in patients with IBD. The most common method for analysis of calprotectin concentration is ELISA. This method is time-consuming and many manufactures have therefore developed rapid tests as a faster alternative for quantification of calprotectin in stool. Aim The aim of the study was to evaluate one ELISA and one rapid test from the same manufacture compare the data with the existing ELISA-method used in the laboratory for routine analysis. Methods A rapid test (CalFast) and an ELISA method (CalPrest) from Eurospital, were used for analysis of calprotectin in stool. These two methods were compared with known concentrations of calprotectin obtained by the ELISA method from Bühlmann used in the routine work.  Results The results showed poor correlation between the rapid test and the ELISA method. Furthermore, the comparison between the two ELISA-methods showed a poor correlation. Conclusion Evaluation of the two new methods showed poor correlation with the existing ELISA method from Bühlmann. Evaluation of the rapid test did not show any correlation with the two ELISA methods and the data cannot be trusted. It is difficult to conclude which of the two ELISA methods gives accurate results due to the absence of an international standard.

Background Calprotectin is a protein expressed in the cytoplasm inside the neutrophile granulocytes. During inflammatory bowel disease (IBD), the neutrophile granulocytes are involved in a complex interaction at the inflammatory area where they die and release their content into the intestinal lumen. Therefore, calprotectin in stool is a suitable marker for diagnosis and measurement of the disease-activity in patients with IBD. The most commonly used method to detect calprotectin in stool is ELISA, but the process of manual preparation of stool samples is time-consuming. Aim The objective of the study was to evaluate an extraction method that could replace manual preparation of fecal samples and to compare different methods for measuring Calprotectin in stool using two ELISA-methods from two manufacturers and one rapidtest. Methods For extraction of calprotectin from stool samples we used sample collector tubes from Epitope Diagnostics and fecal preparation kits from Roche. Two different ELISA-kits for measuring calprotectin concentration in stool were compared. Measurements of calprotectin with rapid-test from Epitope Diagnostics were also performed and were compared with the two ELISA kits. Results The results indicate a poor correlation between two extraction methods with Sample Collector Tube and Roche preparation kit. The comparison between the two ELISA-kits showed poor correlation. Evaluation of rapid test showed 33% false negative results with a cut-off value at 50 mg/kg. Conclusion Evaluation of products from Epitope Diagnostics showed poor correlation with the Bühlmann ELISA and an unreliable rapid test. Therefore, none of evaluated products from Epitope Diagnostics is accurate enough to be used for clinical diagnosis in the laboratory.

Thesis (M. Sc.) Uppsala : Sveriges lantbruksuniv., 2006.

A criptococose é uma doença invasiva capaz de apresentar-se de forma fatal podendo acometer pacientes imunocompetentes e imunocomprometidos. Os agentes etiológicos Cryptococcus neoformans var. grubii e C. neoformans var. neoformans apresentam distribuição cosmopolita, sendo as excretas de pombos o seu principal reservatório. Com o advento de terapias imunossupressoras e a pandemia de HIV, observou-se o aumento significativo de casos de pacientes com criptococose. Atualmente, o diagnóstico é baseado na apresentação clínica, na observação microscópica de líquor corado com tinta da Índia e/ou no isolamento em cultura. Neste trabalho desenvolveu-se um ELISA para detecção de anticorpos contra C. neoformans var. grubii em soro de pacientes utilizando como antígeno um extrato protéico total de uma linhagem clínica isolada de um paciente com criptococose (HC6). Foram testados através de ELISA 40 amostras de soros de pacientes com criptococose, sendo 67,5% positivos e 32,5% falsos negativos. Como controles negativos foram testados 82 amostras de soros de indivíduos hígidos, dos quais 26,82% apresentaram resultados positivos para os testes realizados. Para testar a reatividade cruzada, foram utilizadas 10 amostras de pacientes com histoplasmose (20% de reatividade cruzada), 9 amostras de pacientes com paracoccidioidomicose (66,6% de reatividade cruzada), 9 amostras de pacientes com candidose (13,3% de reatividade cruzada) e 7 amostras de pacientes com aspergilose (14,28% de reatividade cruzada). Visando solucionar o problema da reatividade cruzada, identificamos proteínas antigênicas de C. neoformans var. grubii por eletroforese bidimensional seguida por western blot e espectrometria de massa (MALDI-TOF MS). Das 75 amostras analisadas, quatro foram identificadas: uma proteína hipotética, 2 isoformas de HSPs 70 e uma catalase-2. As proteínas identificadas apresentaram baixa similaridade com ortólogas de outros fungos patogênicos, sendo, dessa forma, possíveis alvos para a padronização do ELISA e diagnóstico da criptococose. / Cryptococosis is an invasive and potentially fatal disease. Cryptococcus neoformans is the etiological agent, which can affect both immunocompromised and immunocompetent individuals. C. neoformans var. grubii and C. neoformans var. neoformans are cosmopolitan and their major natural reservoir is the excrement from pigeons. With the advent of immunosupressor therapies and the pandemic HIV infection, a significant augmentation of cryptococosis cases in humans was observed. Nowadays cryptococcosis diagnosis is based on the clinical presentation, India ink sample preparation methods and/or in vitro culture isolation. In this work we had developed an ELISA to detect antibodies against C. neoformans var. grubii in serum from patients with cryptococcosis using as antigens a whole cell protein extract from a clinical cell line isolate (HC6). Sera from 40 patients with cryptococcosis were tested by ELISA. From these, 67.5% were positives and 32.5% were false-negatives. As a negative control 82 samples from health subjects were also tested, from these 26.82% were positives. To test cross-reactivity, samples from 10 patients with histoplasmosis (20% cross-reactivity), 9 from patients with paracoccidioidomicosis (66.6% cross-reactivity), 9 from patients with candidosis (13.3% cross-reactivity) and 7 from patients with aspergilosis (14.28% cross-reactivity) were tested. To solve the cross-reactivity problem, we searched immunogenic proteins which were specific to C. neoformans var. grubii applying two-dimensional polyacrylamide gel electrophoresis (2DE-PAGE) followed by western blot and mass spectrometry (MALDI-TOF MS). From the 75 sample analyzed, four were identified: one as a hypothetic protein, two HSPs 70 isoforms and the protein catalase 2. These proteins showed low similarity with orthologues from other pathogenic fungi, and are potential targets to further of the standardizing cryptococosis diagnosis by ELISA.