[en] Human toxocariasis (HT) is a cosmopolitan zoonotic disease caused by the migration of the larval stage of the roundworm Toxocara canis. Current HT diagnostic methods do not discriminate between active and past infections. Here, we present a method to quantify Toxocara excretory/secretory antigen, aiming to identify active cases of HT. High specificity is achieved by employing nanobodies (Nbs), single domain antigen binding fragments from camelid heavy chain-only antibodies. High sensitivity is obtained by the design of an electrochemical magnetosensor with an amperometric read-out. Reliable detection of TES antigen at 10 and 30 pg/mL level was demonstrated in phosphate buffered saline and serum, respectively. Moreover, the assay showed no cross-reactivity with other nematode antigens. To our knowledge, this is the most sensitive method to quantify the TES antigen so far. It also has great potential to develop point of care diagnostic systems in other conditions where high sensitivity and specificity are required.
Disciplines :
Biotechnology
Author, co-author :
Morales Yánez, Francisco Javier ; Vrije Universiteit Brussel - VUB > Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium
Trashin, Stanislav; Universiteit Antwerpen - UA > Department of Chemistry > AXES Research Group
Hermy, Marie; Institute of Tropical Medicine Antwerp > Department of Biomedical Sciences > Unit of Medical Helminthology
Sariego, Idalia; Institute of Tropical Medicine Pedro Kouri > Department of Parasitology
Polman, Katja; Institute of Tropical Medicine Antwerp > Department of Biomedical Sciences > Unit of Medical Helminthology
Glickman, L. T.; Schantz, P. M. Epidemiology and pathogenesis of zoonotic toxocariasis. Epidemiol. Rev. 1981, 3, 230-250, 10.1093/oxfordjournals.epirev.a036235
Magnaval, J. F.; Michault, A.; Calon, N.; Charlet, J. P. Epidemiology of human toxocariasis in La Reúnion. Trans. R. Soc. Trop. Med. Hyg. 1994, 88, 531-533, 10.1016/0035-9203(94)90148-1
Schoenardie, E. R.; Scaini, C. J.; Brod, C. S.; Pepe, M. S.; Villela, M. M.; McBride, A. J.; Borsuk, S.; Berne, M. E. Seroprevalence of Toxocara infection in children from southern Brazil. J. Parasitol. 2013, 99, 537-539, 10.1645/GE-3182
Alvarado-Esquivel, C.; Hernandez-Tinoco, J.; Sanchez-Anguiano, L. F. Seroepidemiology of Toxocara infection in patients with vision impairment and blindness in durango, Mexico. J. Clin. Med. Res. 2015, 7, 176-181, 10.14740/jocmr2032w
Stensvold, C. R.; Skov, J.; Moller, L. N.; Jensen, P. M.; Kapel, C. M.; Petersen, E.; Nielsen, H. V. Seroprevalence of human toxocariasis in Denmark. Clin. Vaccine Immunol. 2009, 16, 1372-1373, 10.1128/CVI.00234-09
Buijs, J.; Borsboom, G.; van Gemund, J. J.; Hazebroek, A.; van Dongen, P. A.; van Knapen, F.; Neijens, H. J. Toxocara seroprevalence in 5-year-old elementary schoolchildren: Relation with allergic asthma. Am. J. Epidemiol. 1994, 140, 839-847, 10.1093/oxfordjournals.aje.a117332
Nijsse, R.; Ploeger, H. W.; Wagenaar, J. A.; Mughini-Gras, L. Toxocara canis in household dogs: Prevalence, risk factors and owners' attitude towards deworming. Parasitol. Res. 2015, 114, 561-569, 10.1007/s00436-014-4218-9
Mughini-Gras, L.; Harms, M.; van Pelt, W.; Pinelli, E.; Kortbeek, T. Seroepidemiology of human Toxocara and Ascaris infections in the Netherlands. Parasitol. Res. 2016, 115, 3779-3794, 10.1007/s00436-016-5139-6
Fillaux, J.; Magnaval, J. F. Laboratory diagnosis of human toxocariasis. Vet. Parasitol. 2013, 193, 327-336, 10.1016/j.vetpar.2012.12.028
Robertson, B. D.; Burkot, T. R.; Gillespie, S. H.; Kennedy, M. W.; Wambai, Z.; Maizels, R. M. Detection of circulating parasite antigen and specific antibody in Toxocara canis infections. Clin. Exp. Immunol. 1988, 74, 236-241
Ishiyamna, S.; Ono, K.; Rai, S. K.; Uga, S. Method for detecting circulating Toxocara canis antigen and its application in human serum samples. Nepal Med. Coll. J. 2009, 11, 9-13
Rodriguez-Caballero, A.; Martinez-Gordillo, M. N.; Medina-Flores, Y.; Medina-Escutia, M. E.; Meza-Lucas, A.; Correa, D.; Caballero-Salazar, S.; Ponce-Macotela, M. Successful capture of Toxocara canis larva antigens from human serum samples. Parasites Vectors 2015, 8, 264-230, 10.1186/s13071-015-0875-5
Voskuil, J. Commercial antibodies and their validation. F1000Research 2014, 3, 232, 10.12688/f1000research.4966.2
Schumacher, D.; Helma, J.; Schneider, A. F. L.; Leonhardt, H.; Hackenberger, C. P. R. Nanobodies: Chemical functionalization strategies and intracellular applications. Angew. Chem., Int. Ed. 2018, 57, 2314-2333, 10.1002/anie.201708459
Morales-Yanez, F. J.; Sariego, I.; Vincke, C.; Hassanzadeh-Ghassabeh, G.; Polman, K.; Muyldermans, S. An innovative approach in the detection of Toxocara canis excretory/secretory antigens using specific nanobodies. Int. J. Parasitol. 2019, 49, 635-645, 10.1016/j.ijpara.2019.03.004
Goodsell, D. S. Nanobodies. RCSB Protein Data Bank 2011, na, 10.2210/rcsb_pdb/mom_2011_4
Zacco, E.; Pividori, M. I.; Alegret, S.; Galve, R.; Marco, M. P. Electrochemical magnetoimmunosensing strategy for the detection of pesticides residues. Anal. Chem. 2006, 78, 1780-8, 10.1021/ac0512610
Zacco, E.; Adrian, J.; Galve, R.; Marco, M. P.; Alegret, S.; Pividori, M. I. Electrochemical magneto immunosensing of antibiotic residues in milk. Biosens. Bioelectron. 2007, 22, 2184-2191, 10.1016/j.bios.2006.10.014
Richardson, J.; Hawkins, P.; Luxton, R. The use of coated paramagnetic particles as a physical label in a magneto-immunoassay. Biosens. Bioelectron. 2001, 16, 989-993, 10.1016/S0956-5663(01)00201-9
Croci, L.; Delibato, E.; Volpe, G.; Palleschi, G. A Rapid Electrochemical Elisa for the Detection of Salmonella in Meat Samples. Anal. Lett. 2001, 34, 2597-2607, 10.1081/AL-100108407
Campuzano, S.; Yáñez-Sedenõ, P.; Pingarrón, J. M. Electrochemical Biosensing for the Diagnosis of Viral Infections and Tropical Diseases. ChemElectroChem 2017, 4, 753-777, 10.1002/celc.201600805
de Souza Castilho, M.; Laube, T.; Yamanaka, H.; Alegret, S.; Pividori, M. I. Magneto immunoassays for Plasmodium falciparum histidine-rich protein 2 related to malaria based on magnetic nanoparticles. Anal. Chem. 2011, 83, 5570-7, 10.1021/ac200573s
Ferreira, A. A.; Colli, W.; da Costa, P. I.; Yamanaka, H. Immunosensor for the diagnosis of Chagas' disease. Biosens. Bioelectron. 2005, 21, 175-181, 10.1016/j.bios.2004.08.001
Diaz-Gonzalez, M.; Gonzalez-Garcia, M. B.; Costa-Garcia, A. Immunosensor for Mycobacterium tuberculosis on screen-printed carbon electrodes. Biosens. Bioelectron. 2005, 20, 2035-2043, 10.1016/j.bios.2004.09.035
Mach, K. E.; Mohan, R.; Patel, S.; Wong, P. K.; Hsieh, M.; Liao, J. C. Development of a Biosensor-Based Rapid Urine Test for Detection of Urogenital Schistosomiasis. PLoS Neglected Trop. Dis. 2015, 9, e0003845, 10.1371/journal.pntd.0003845
de Savigny, D. H. In vitro maintenance of Toxocara canis larvae and a simple method for the production of Toxocara ES antigen for use in serodiagnostic tests for visceral larva migrans. J. Parasitol. 1975, 61, 781, 10.2307/3279492
Thomas, D.; Jeyathilakan, N.; Abdul Basith, S.; Senthilkumar, T. M. In vitro production of Toxocara canis excretory-secretory (TES) antigen. J. Parasit. Dis. 2016, 40, 1038-1043, 10.1007/s12639-014-0630-4
Kennedy, W. M. Toxocara: The Enigmatic Parasite; CABI Publishing: Oxfordshire, 2006.
Conrath, K. E.; Lauwereys, M.; Galleni, M.; Matagne, A.; Frere, J. M.; Kinne, J.; Wyns, L.; Muyldermans, S. b-Lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae. Antimicrob. Agents Chemother. 2001, 45, 2807-2812, 10.1128/AAC.45.10.2807-2812.2001
Vincke, C.; Gutiérrez, C.; Wernery, U.; Devoogdt, N.; Hassanzadeh-Ghassabeh, G.; Muyldermans, S. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs. In Antibody Engineering. Methods in Molecular Biology (Methods and Protocols); Humana Press: Totowa, 2012; Vol. 907, pp 145-176.
Pardon, E.; Laeremans, T.; Triest, S.; Rasmussen, S. G.; Wohlkonig, A.; Ruf, A.; Muyldermans, S.; Hol, W. G.; Kobilka, B. K.; Steyaert, J. A general protocol for the generation of Nanobodies for structural biology. Nat. Protoc. 2014, 9, 674-693, 10.1038/nprot.2014.039
Beckett, D.; Kovaleva, E.; Schatz, P. J. A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci. 1999, 8, 921-929, 10.1110/ps.8.4.921
Rusling, J. F.; Kumar, C. V.; Gutkind, J. S.; Patel, V. Measurement of biomarker proteins for point-of-care early detection and monitoring of cancer. Analyst 2010, 135, 2496-2511, 10.1039/c0an00204f
Bos, E. S.; van der Doelen, A. A.; Rooy, N. v.; Schuurs, A. H. W. M. 3,3′,5,5′-Tetramethylbenzidine as an Ames Test Negative Chromogen for Horse-Radish Peroxidase in Enzyme-Immunoassay. J. Immunoassay 1981, 2, 187-204, 10.1080/15321818108056977
Even-Desrumeaux, K.; Fourquet, P.; Secq, V.; Baty, D.; Chames, P. Single-domain antibodies: A versatile and rich source of binders for breast cancer diagnostic approaches. Mol. BioSyst. 2012, 8, 2385-2394, 10.1039/c2mb25063b
Rodriguez-Caballero, A.; Martinez-Gordillo, M. N.; Caballero-Salazar, S.; Rufino-Gonzalez, Y.; Ponce-Macotela, M. Toxocara canis: Analysis of the kinetics of antigen release and antibody production in an in vivo model for the detection of past or present infection. Vet. Parasitol. 2017, 243, 183-187, 10.1016/j.vetpar.2017.06.027
Badley, J. E.; Grieve, R. B.; Bowman, D. D.; Glickman, L. T.; Rockey, J. H. Analysis of toxocara canis larval excretory-secretory antigens: Physicochemical characterization and antibody recognition. J. Parasitol. 1987, 73, 593, 10.2307/3282142
Hall, J. E. Guyton and Hall Textbook of Medical Physiology, 13 th ed.; Saunders: Philadelphia, 2016.
de Savigny, D. H.; Tizard, I. R. Toxocaral larva migrans: The use of larval secretory antigens in haemagglutination and soluble antigen fluorescent antibody tests. Trans. R. Soc. Trop. Med. Hyg. 1977, 71, 501-507, 10.1016/0035-9203(77)90144-4
Bowman, D. D.; Mika-Grieve, M.; Grieve, R. B. Circulating excretory-secretory antigen levels and specific antibody responses in mice infected with Toxocara canis. Am. J. Trop. Med. Hyg. 1987, 36, 75-82, 10.4269/ajtmh.1987.36.75
Niel, G.; Pinon, J. M.; Couture, J.; Rosin, G.; Gentilini, M. Aspect immunologique de syndromes hypereósinophiliques évoquant une Larva migrans viscérale. Bull. Soc. Pathol. Exot. Ses Fil. 1973, 66, 324-332
Magnaval, J. F.; Glickman, L. T.; Grieve, R. B.; Carriere, E. D.; Dubly, E. Intérêt de l'immunoélectrophorèse utilisant des extraits d'organes génitaux d'Ascaris suum dans le diagnostic de la toxocarose. Bull. Soc. Pathol. Exot. Ses Fil. 1986, 79, 634-641
