Immune thrombocytopenia (ITP) is an autoimmune hematologic disorder characterized by a low platelet count due to increased platelet destruction or decreased production. In primary ITP, the patient can have a low platelet count (<100 billion cells/L) for clinically unknown reasons. ITP is a rare disease that affects approximately 3/100 000 adults each year and some patients may experience bleeding symptoms. Autoantibody-mediated autoimmunity plays a role in the destruction of platelets by targeting platelet glycoproteins (GPs). Autoantibodies against platelet membrane GPIIbIIIa and GPIbIX are observed in about 50% of patients through direct antigen-capture assays, and 18% in patients through indirect antigen-capture assays. It is possible that some antibodies may not be detectable due to affinity or titre, or there may be other factors involved in platelet destruction. Currently, there is no definitive diagnostic test available for ITP, as a result of low assay sensitivity and different mechanisms involved in disease pathogenesis. The objective of this study was to use a novel approach to increase autoantibody detection unique to ITP patients. Total IgG was purified from patient and control plasma samples. A streptavidin-based antigen-capture assay was optimized to test the effect of biotinylation on the detection of anti-GPIIbIIIa and anti-GPIbIX autoantibodies in primary ITP patients (n=14), secondary ITP patients (n=3), non-immune thrombocytopenic controls (n=2) and healthy controls (n=16). Streptavidin-coated biosensors were used in an optimized biolayer interferometry (BLI) assay to study autoantibodies binding to biotinylated GPIIbIIIa and GPIbIX. Detection of anti-GPIIbIIIa autoantibodies in the streptavidin antigen-capture assay had a sensitivity of 24% and anti-GPIbIX autoantibodies had a sensitivity of 25%. BLI showed binding of autoantibodies in approximately 5% of ITP samples for both GPIIbIIIa and GPIbIX. The samples that had detectable autoantibodies in the antigen-capture assay did not have detectable antibodies in the BLI assay. BLI was not able to confirm antibody detection found in enzyme immunoassays. / Thesis / Master of Science (MSc) / Platelets are blood cells involved in clotting at sites of injury. Immune thrombocytopenia (ITP) is a disease defined by a low platelet count that can lead to bleeding. ITP is a rare disease that affects 3 in 100 000 adults every year. ITP is thought to be caused by proteins known as antibodies that bind self-platelets and lead to their destruction. These antibodies are directly found on approximately 50% of patients’ platelets, and only 18% of patients have antibodies in circulation. It is possible in many patients, antibodies are present at a low concentration, or are too weak to be detected in antibody tests. In this study, a new technology known as biolayer interferometry was employed to find antibodies in a higher percentage of patients. Results showed only 6% of ITP patients had detectable antibodies in their circulation. This research will improve our understanding of antibodies in ITP.

En dépit d’importants progrès dans le domaine de la thérapie anti-cancéreuse, les traitements actuels restent controversés, notamment en raison de la quantité importante d'effets secondaires induits. L'immunothérapie ciblée a récemment émergée en tant qu'alternative, afin d'améliorer les modalités de traitement des patients atteints du cancer. Malgré tout, seul un nombre limité d’approches sont aujourd’hui disponibles, et une grande partie des problèmes demeurent actuellement sans solution. C'est dans ce contexte que nous nous sommes intéressés à la conception de structures biomoléculaires innovantes et bifonctionnelles, capables de rediriger des anticorps endogènes, présents naturellement dans la circulation sanguine de l'homme, contre les tumeurs et, ce, sans immunisation préalable. Les anticorps naturels circulant étant polyspécifiques et ayant la capacité d’interagir avec des antigènes glycosylés, nous nous sommes plus particulièrement concentrés sur la conception de glycoconjugués multivalents, ligands d’anticorps endogènes. Une première partie de notre étude a consisté à synthétiser différents glycodendrimères multivalents, reposant sur des châssis peptidiques et obtenus par ligations chimiosélectives, tout en variant la nature du motif glycosylé et des plateformes, ainsi que la valence du conjugué. Puis, dans un second temps, des tests d’interaction par biopuce ont été mis en place avec une lectine modèle, la lectine Helix Pomatia Agglutinin (HPA). Des protocoles expérimentaux visant à calculer des constantes de dissociation de surface, ainsi que des IC50 ont été mis en place, permettant d’identifier de bons ligands de HPA avec des affinités de l’ordre du nanomolaire. Les tests par biopuce ont ensuite été confirmés avec d’autres méthodes d’analyses (BLI, ELLA). Finalement, afin d'identifier des architectures tri-dimensionnelles permettant une affinité optimale avec des anticorps, les tests d’interaction ont été adaptés au criblage de séra humains. Un large panel de glycoconjugués a alors été criblé par biopuce avec une vingtaine de séra, permettant la détermination de structures glycosylés prometteuses, qui pourront par la suite être utilisées dans le cadre de notre approche anti-cancéreuse. / Despite significant progress in anti-cancer therapy, current treatments are still controversial due to numerous side effects. Targeted immunotherapy recently emerged as an ideal alternative to improve treatment modalities for cancer patients. However, very limited approaches are available today and major issues remain to be addressed. In this context, we are interested in the design of biomolecular structures, innovative and bifunctional, able to hijack endogenous antibodies - which are naturally present in the human blood stream - toward cancer cells without pre-immunisation. Since natural circulating antibodies are polyspecific and have the ability to interact with multiple carbohydrate antigens, we focused on the design of multivalent glycodendrimers, as ligands for endogenous antibodies. The first part of our study consisted in synthesizing several multivalent glycoconjugates, based on peptide scaffolds and obtained by chemoselective ligations. To evaluate their influence on antibodies, the nature of both the carbohydrate and the scaffold, and the valency were varied. Then, in a second part of the study, microarray assays were developed with a model lectin, the Helix Pomatia Agglutinin (HPA). Experimental procedures were designed to determine surface dissociation constant and IC50 values, leading to the identification of high affinity ligands for HPA in the nanomolar range. Microarray assays were confirmed by other analytical methods (BLI, ELLA). Finally, the assays on slides were adapted to human sera screening, in order to identify tridimensional architectures highly affine to sera antibodies. A large panel of glycoconjugates were screened by microarray with around twenty sera, leading to the determination of promising glycosylated structures, as antibody ligands. The latter could be subsequently used for our anti-cancer approach.