Development and evaluation of a sensitive and specific assay for diagnosis of human toxocariasis by use of three recombinant antigens (TES-26, TES-30USM, and TES-120) - PubMed (original) (raw)

Development and evaluation of a sensitive and specific assay for diagnosis of human toxocariasis by use of three recombinant antigens (TES-26, TES-30USM, and TES-120)

Suharni Mohamad et al. J Clin Microbiol. 2009 Jun.

Abstract

Diagnosis of human toxocariasis currently relies on serologic tests that use Toxocara excretory-secretory (TES) antigen to detect immunoglobulin G (IgG) antibodies to the larvae. In general, however, these assays do not have adequate specificity for use in countries in which other soil-transmitted helminths are endemic. The use of recombinant antigens in these assays, however, is promising for improving the specificity of the diagnosis of toxocariasis. Toward this goal, we developed an IgG4 enzyme-linked immunosorbent assay (ELISA) involving three recombinant antigens: rTES-30USM (previously produced), rTES-26, and rTES-120. The latter two antigens were produced by reverse transcription-PCR cloning; subcloned into glutathione S-transferase (GST)-tagged and His-tagged prokaryotic expression vectors, respectively; and expressed in Escherichia coli. The recombinant proteins were subsequently purified by affinity chromatography using GST and His-Trap resins. The diagnostic potential of each purified recombinant antigen was tested with various immunoglobulin classes (IgG, IgM, and IgE) and IgG subclasses. The IgG4 ELISA was determined to have the highest specificity and was further evaluated using a panel of serum samples. The rTES-26 IgG4 ELISA showed 80.0% (24/30 samples positive) sensitivity, and both the rTES-30USM IgG4 ELISA and rTES-120 IgG4 ELISA had 93.0% (28/30) sensitivity. Combined use of rTES-120 and rTES-30 IgG4 ELISA for the diagnosis of toxocariasis provided 100% sensitivity. The specificities of rTES-26, rTES-30USM, and rTES-120 antigens were 96.2%, 93.9%, and 92.0%, respectively. These results indicate that the development of a diagnostic test using the three recombinant antigens will allow for more-accurate detection of toxocariasis.

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Figures

FIG. 1.

FIG. 1.

Agarose gel electrophoresis of the amplified RT-PCR products of genes encoding TES-26 and TES-120. Lane 1, 100-bp DNA ladder; lane 2, amplified product of genes encoding TES-26; lane 3, amplified product of genes encoding TES-120; lane 4, positive control; lane 5, negative control.

FIG. 2.

FIG. 2.

SDS-PAGE of the purified TES-26 GST fusion and TES-120 His-tagged proteins. Lane 1, protein marker; lane 2, rTES-120 antigen (26 kDa); lane 3, rTES-26 antigen (72 kDa).

FIG. 3.

FIG. 3.

(a) Western blot analysis of rTES-26 antigen probed with various categories of infected and noninfected serum. Lane 1, protein marker; lanes 2, 3, and 4, sera from three different _Toxocara_-infected patients; lane 5, serum from trichuriasis patient; lane 6, serum from toxoplasmosis patient; lanes 7 and 8, sera from apparently healthy people. (b) Western blot analysis of rTES-120 antigen probed with sera from variously infected and healthy individuals. Lane 1, protein marker; lanes 2, 3, and 4, sera from three different _Toxocara_-infected patients; lane 5, serum from trichuriasis patient; lane 6, serum from toxoplasmosis patient; lanes 7 and 8, sera from apparently healthy people.

References

    1. Chung, L. Y., B. H. Fang, J. H. Chang, S. M. Chye, and C. M. Yen. 2004. The infectivity and antigenicity of Toxocara canis eggs can be retained after long-term preservation. Ann. Trop. Med. Parasitol. 98251-260. - PubMed
    1. Despommier, D. 2003. Toxocariasis: clinical aspects, epidemiology, medical ecology, and molecular aspects. Clin. Microbiol. Rev. 16265-272. - PMC - PubMed
    1. Fong, M. Y., and Y. L. Lau. 2004. Recombinant expression of the larval excretory-secretory antigen TES-120 of Toxocara canis in the methylotropic yeast Pichia pastoris. Parasitol. Res. 92173-176. - PubMed
    1. Fong, M. Y., Y. L. Lau, I. Init, I. Jamaiah, A. K. Anuar, and N. Rahmah. 2003. Recombinant expression of Toxocara canis excretory-secretory antigens TES-120 in Escherichia coli. Southeast Asian J. Trop. Med. Public Health 34723-726. - PubMed
    1. Gems, D., C. J. Ferguson, B. D. Robertson, R. Nieves, A. P. Page, M. L. Blaxter, and R. M. Maizels. 1995. An abundant, trans-spliced mRNA from Toxocara canis infective larvae encodes a 26-kDa protein with homology to phosphatidylethanolamine-binding proteins. J. Biol. Chem. 27018517-18522. - PubMed

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