Development of innovative nanobody-based strategies to improve the diagnosis of human toxocariasis

Human toxocariasis (HT) is a zoonotic disease caused by the infection of the larval stage of the dog roundworm Toxocara canis. Detection of active HT infections is elusive due to the lack of sensitivity and specificity of current laboratory tools. In this thesis, we introduce a sensitive and specific nanobody-based sandwich ELISA to detect the T. canis excretory-secretory antigen (TES) with a limit of detection of 0.650 ng/ml in serum spiked with TES. This ELISA employs bivalent biotinylated nanobodies as capturing agent and nanobodies chemically coupled to horseradish peroxidase for detection. Higher sensitivity was achieved converting the ELISA into an electrochemical magnetosensor assay, obtaining a limit 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% (33/87) sera were positive for TES. Positivity was significantly correlated with eosinophils >19% of total white blood cell count. Additionally, the test showed low cross-reactivity to other helminths. This is to our knowledge, the most sensitive and specific immunoassay to diagnose HT currently available. This research highlights the impressive versatility of nanobodies for the development of highly specific and sensitive immunoassays.