Human toxocariasis (HT) is a zoonotic disease caused by the infection of the larval stage of the dog roundworm Toxocara canis. Currently, laboratory diagnosis of HT relies on the ELISA-based detection of specific immunoglobulins against the T. canis excretory/secretory (TES) antigen (Ab TES-ELISA). TES is a mix of highly glycosylated proteins that the parasite releases into the tissues during the migration of larval stages in the human host. The most important drawback of the Ab TES-ELISA is its inability to distinguish between active and past infections. Moreover, cross-reactivity with other helminths is frequent. This is particularly important in tropical regions, where polyparasitism is frequent. In this PhD thesis, we introduce a sensitive and specific nanobody-based sandwich ELISA (Nb-ELISA) to detect TES antigen with a limit of detection of 0.650 ng/ml in serum spiked with TES. This Nb-ELISA employs bivalent biotinylated nanobodies as capturing agent and nanobodies chemically coupled to horseradish peroxidase for detection. The Nb-ELISA was able to detect TES in mice sera taken 3 days after they were experimentally infected with T. canis eggs. Even higher sensitivity was achieved by converting the ELISA into an electrochemical magnetosensor assay, whereby the reaction takes place on the surface of streptavidin-precoated paramagnetic beads. In this configuration, the assay had a limit of detection of 10 pg/ml in serum spiked with TES. This assay was evaluated in serum samples from children of remote rural communities in the province of Esmeraldas (Ecuador). We found that 38% (33/84) of the sera were positive for TES antigen. Positivity was significantly correlated with eosinophilia. Additionally, the test showed no cross-reactivity with other helminths. To our knowledge, this is the most sensitive and specific immunoassay to diagnose HT currently available. As the electrochemical assay provides evidence of active Toxocara infections, it has great potential to significantly improve HT diagnosis. It also provides opportunities to develop point of care diagnostic systems for other diseases where high sensitivity and specificity are required. This research highlights the impressive versatility of nanobodies for the development of innovative immunoassays.
|Qualification||Doctor of Philosophy|
|Place of Publication||Brussel|
|Publication status||Published - 2019|