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Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26

Authors :
Jan Pohl
Isabel McAuliffe
Sukwan Handali
Keith Levert
Matthew S. Reed
Bin Zhan
Hilda N. Rivera
Ryan E. Wiegand
Holly M. Chastain
Peter J. Hotez
John P. Anderson
Patricia P. Wilkins
Lisa N. Rascoe
Source :
PLoS Neglected Tropical Diseases, Vol 9, Iss 10, p e0004168 (2015), PLoS Neglected Tropical Diseases
Publication Year :
2015
Publisher :
Public Library of Science (PLoS), 2015.

Abstract

The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag.<br />Author Summary The roundworms Toxocara canis and Toxocara cati cause a broad spectrum of clinical disease in humans. Children are at particular risk of toxocariasis when they play in areas potentially contaminated with Toxocara eggs, such as playgrounds or sandboxes and ingest embryonated roundworm eggs. Currently, diagnosis for toxocariasis relies on clinical signs, history of exposure to puppies or kittens, laboratory findings (including eosinophilia), and the detection of antibodies to Toxocara antigens. The enzyme immunoassay using T. canis excretory secretory antigens from infective-stage larvae is the most useful diagnostic test for toxocaral visceral larva migrans (VLM) and ocular larva migrans (OLM) and is the preferred assay used by most laboratories in the U.S. and worldwide. Although the EIA has been robust and reliable, improvement should be made in the specificity of the assay and the availability of a consistent antigen source. The crude TES-Ag shows cross-reactivity with antibodies from other common helminth infections of humans which reduces the usefulness of native, unfractionated TES Ag-based serodiagnosis in regions where poly-parasitism is endemic. To improve the assay performance, target antigenic proteins from T. canis excretory secretory antigens were identified using 2D gel electrophoresis. Three antigenic proteins sequences were found, expressed, and developed into Luminex bead-based assays. The combined use of two recombinant antigens (Tc-CTL-1 and Tc-TES-26) represents an improvement over the existing immunodiagnostic methods that rely on native parasite materials. In the future, additional antigens could be added from other parasites that cause larval migrans to form a single method for detecting larval migrans syndromes.

Details

Language :
English
ISSN :
19352735 and 19352727
Volume :
9
Issue :
10
Database :
OpenAIRE
Journal :
PLoS Neglected Tropical Diseases
Accession number :
edsair.doi.dedup.....76df1571dbdb519cac11e1007b637394