An innovative approach in the detection of Toxocara canis excretory/secretory antigens using specific nanobodies

Francisco J. Morales-Yanez*, Idalia Sariego, Cécile Vincke, Gholamreza Hassanzadeh-Ghassabeh, Katja Polman, Serge Muyldermans

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

Human toxocariasis is a zoonosis resulting from the migration of larval stages of the dog parasite Toxocara canis into the human paratenic host. Despite its well-known limitations, serology remains the most important tool to diagnose the disease. Our objective was to employ camelid single domain antibody fragments also known as nanobodies (Nbs) for a specific and sensitive detection of Toxocara canis excretory/secretory (TES) antigens. From an alpaca immune Nb library, we retrieved different Nbs with specificity for TES antigens. Based on ELISA experiments, these Nbs did not show any cross-reactivity with Ascaris lumbricoides, Ascaris suum, Pseudoterranova decipiens, Anisakis simplex and Angiostrongylus cantonensis larval antigens. Western blot and immunocapturing revealed that Nbs 1TCE39, 1TCE52 and 2TCE49 recognise shared epitopes on different components of TES antigen. The presence of disulphide bonds in the target antigen seems to be essential for recognition of the epitopes by these three Nbs. Three separate sandwich ELISA formats, using monovalent and bivalent Nbs, were assessed to maximise the detection of TES antigens in solution. The combination of biotinylated, bivalent Nb 2TCE49 on a streptavidin pre-coated plate to capture TES antigens, and Nb 1TCE39 chemically coupled to horseradish peroxidase for detection of the captured TES antigens, yielded the most sensitive ELISA with a limit of detection of 0.650 ng/ml of TES antigen, spiked in serum. Moreover, the assay was able to detect TES antigens in sera from mice, taken 3 days after the animals were experimentally infected with T. canis. The specific characteristics of Nbs make this ELISA not only a promising tool for the detection of TES antigens in clinical samples, but also for a detailed structural and functional study of TES antigens.

Original languageEnglish
Pages (from-to)635-645
Number of pages11
JournalInternational Journal for Parasitology
Volume49
Issue number8
DOIs
Publication statusPublished - 1 Jul 2019

Funding

This project was funded by the Fonds Wetenschappelijk Onderzoek-Vlaanderen (FWO-Vlaanderen, Belgium), project No. G.0189.13N. The authors would like to thank Ema Romão for fruitful conversations regarding nanobody cloning and production as well as Natalia Smiejkowska, Vaiva Gaspariunaite, Marie Hermy, Jan Van Gompel, Anke Van Hul and Koen de Witte for their excellent technical assistance in the laboratory. Our special thanks to Prof. Pierre Dorny (Institute of Tropical Medicine Antwerp, Belgium) and Dr. Beatrice Nickel (Swiss Institute of Tropical Medicine, Switzerland) for providing the battery of antigens needed for cross-reactivity testing. Thanks also to Clémentine Roucher and Linda Paredis for their critical review of the paper. The authors declare that they have no competing interests. Appendix A

FundersFunder number
Dr. Beatrice Nickel
Fonds Wetenschappelijk Onderzoek-Vlaanderen
Swiss Institute of Tropical Medicine
Fonds Wetenschappelijk Onderzoek
Vlaamse regering

    Keywords

    • Excretory–secretory antigens
    • Nanobody
    • Sandwich ELISA
    • Single domain antibodies
    • Toxocara canis

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