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Recombinant antigens for immunodiagnosis of cystic echinococcosis.

ABSTRACT: Three cDNAs, termed EpC1, TPxEg and EgG5, were isolated by immunoscreening from an Echinococcus granulosus cDNA library. The recombinant phages exhibited strong reactivity with sera from humans with confirmed cystic echinococcosis (CE) and with sera from mice infected with E. granulosus oncospheres. The cDNAs were subcloned into a pET vector, expressed as fusion proteins tagged with GST and affinity purified against the GST tag. Of the three recombinant proteins, EpC1 achieved the highest performance for serodiagnosis of CE in Western blot analysis using a panel of clinically defined human sera to initially address the sensitivity and specificity of the molecules. The protein yielded an overall sensitivity of 92.2% and specificity of 95.6%, levels unprecedented taking into account the large panel of 896 human sera that were tested. The strategy used may also prove suitable for improved immunodiagnosis of other parasitic infections.

SUBMITTER: Li J

PROVIDER: S-EPMC411165 | biostudies-literature | 2004

REPOSITORIES: biostudies-literature

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Recombinant antigens for immunodiagnosis of cystic echinococcosis.

Li Jun J Zhang Wen-Bao WB McManus Donald P. DP

Biological procedures online 20040510

Three cDNAs, termed EpC1, TPxEg and EgG5, were isolated by immunoscreening from an Echinococcus granulosus cDNA library. The recombinant phages exhibited strong reactivity with sera from humans with confirmed cystic echinococcosis (CE) and with sera from mice infected with E. granulosus oncospheres. The cDNAs were subcloned into a pET vector, expressed as fusion proteins tagged with GST and affinity purified against the GST tag. Of the three recombinant proteins, EpC1 achieved the highest perfor ...[more]

PMID: 15188015

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Project description:The accuracy of screening tests for detecting cystic echinococcosis (CE) in livestock depends on characteristics of the host-parasite interaction and the extent of serological cross-reactivity with other taeniid species. The AgB8 kDa protein is considered to be the most specific native or recombinant antigen for immunodiagnosis of ovine CE. A particular DNA fragment coding for rAgB8/2 was identified, that provides evidence of specific reaction in the serodiagnosis of metacestode infection. We developed and validated an IgG Enzyme Linked Immunosorbent Assay (ELISA) test using a recombinant antigen B sub-unit EgAgB8/2 (rAgB8/2) of Echinoccocus granulosus sensu lato (s.l.) to estimate CE prevalence in sheep. A 273 bp DNA fragment coding for rAgB8/2 was expressed as a fusion protein (∼30 kDa) and purified by affinity chromatography. Evaluation of the analytical and diagnostic performance of the ELISA followed the World Organisation for Animal Health (OIE) manual, including implementation of serum panels from: uninfected lambs (n = 79); experimentally infected (with 2,000 E. granulosus s.l. eggs each) sheep with subsequent evidence of E. granulosus cysts by necropsy (n = 36), and animals carrying other metacestode/trematode infections (n = 20). The latter were used to assess the cross-reactivity of rAgB8/2, with these animals being naturally infected with Taenia hydatigena, Thysanosoma actinioides and/or Fasciola hepatica. EgAgB8/2 showed cross-reaction with only one serum sample from a sheep infected with Ta. hydatigena out of the 20 animals tested. Furthermore, the kinetics of the humoral response over time in five 6-month old sheep, each experimentally infected with 2,000 E. granulosus s.l. eggs, was evaluated up to 49 weeks (approximately one year) post infection (n = 5). The earliest detectable IgG response against rAgB8/2 was observed in sera from two and four sheep, 7 and 14 days after experimental infection, respectively. The highest immune response across all five animals was found 16 to 24 weeks post infection.

Project description:Cystic echinococcosis (CE) is a neglected zoonotic disease caused by Echinococcus granulosus sensu lato. Diagnosis and monitoring of CE rely primarily on imaging while serology is used as a confirmatory test. However, imaging is not always conclusive and currently available serological assays have suboptimal sensitivity and specificity, lack standardization, and are not useful for patients´ follow-up. Seroassays for CE are usually based on hydatid fluid (HF), a complex, variable antigenic mixture, and cross-reactivity exists especially with alveolar echinococcosis. Recombinant proteins based on immunogenic antigens most abundant in HF, such as AgB1, AgB2 and Ag5, have been used to overcome these limitations. None of them so far showed potential to replace HF; however, their performance have been largely tested on a limited number of samples, and comparison of different antigens using the same cohort has been rarely performed. The combination of several immunogenic epitopes in a single recombinant protein could enhance test sensitivity. For the diagnosis and follow-up of patients with CE, we compared the performance of the crude HF, previously described recombinant 2B2t antigen, and GST-tagged version of 2B2t, and novel designed recombinants (GST-Ag5t and the GST-DIPOL chimera containing AgB1, AgBB2 and Ag5 epitopes) by IgG-ELISA format. Samples belong to a retrospective cohort of 253 well-characterized patients with CE, previously described for the evaluation of the 2B2t antigen, 92 patients with alveolar echinococcosis, and 82 healthy donors. The reference standard for CE diagnosis was the presence of a CE lesion as diagnosed by ultrasonography. The highest sensitivity was obtained with HF [86.7%, 95% confidence interval (CI): 81.2-91.0], followed by GST-2B2t (70.0%, 95% CI: 63.1-76.2), 2B2t (65.5%, 95% CI: 58.5-72.0), GST-Ag5t (64.5%, 95% CI: 57.5-71.1) and GST-DIPOL (63.1%, 95% CI: 56.0-69.7). The GST-2B2t had the best specificity (95.8%, 95% CI: 88.3-99.1) and the lowest cross-reactivity (38.7%, 95% CI: 27.6-50.6). Good response to treatment also correlated to negative test results in the GST-2B2t ELISA. While none of the tested recombinant antigen appears suitable to replace HF for the diagnosis of CE, GST-2B2t should be further explored as a confirmation test, based on its high specificity and low cross-reactivity, and for the follow-up after treatment in those patients with positive serology for this antigen.

Dataset Information

Recombinant antigens for immunodiagnosis of cystic echinococcosis.

Publications

Recombinant antigens for immunodiagnosis of cystic echinococcosis.

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