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Description of a nanobody-based competitive immunoassay to detect tsetse fly exposure.

Caljon G, Hussain S, Vermeiren L, Van Den Abbeele J - PLoS Negl Trop Dis (2015)

Bottom Line: We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species.In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts.In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a bottleneck.

View Article: PubMed Central - PubMed

Affiliation: Unit of Veterinary Protozoology, Department of Biomedical Sciences, Institute of Tropical Medicine Antwerp (ITM), Antwerp, Belgium; Unit of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium; Laboratory of Myeloid Cell Immunology, VIB, Brussels, Belgium.

ABSTRACT

Background: Tsetse flies are the main vectors of human and animal African trypanosomes. The Tsal proteins in tsetse fly saliva were previously identified as suitable biomarkers of bite exposure. A new competitive assay was conceived based on nanobody (Nb) technology to ameliorate the detection of anti-Tsal antibodies in mammalian hosts.

Methodology/principal findings: A camelid-derived Nb library was generated against the Glossina morsitans morsitans sialome and exploited to select Tsal specific Nbs. One of the three identified Nb families (family III, TsalNb-05 and TsalNb-11) was found suitable for anti-Tsal antibody detection in a competitive ELISA format. The competitive ELISA was able to detect exposure to a broad range of tsetse species (G. morsitans morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes) and did not cross-react with the other hematophagous insects (Stomoxys calcitrans and Tabanus yao). Using a collection of plasmas from tsetse-exposed pigs, the new test characteristics were compared with those of the previously described G. m. moristans and rTsal1 indirect ELISAs, revealing equally good specificities (> 95%) and positive predictive values (> 98%) but higher negative predictive values and hence increased sensitivity (> 95%) and accuracy (> 95%).

Conclusion/significance: We have developed a highly accurate Nb-based competitive immunoassay to detect specific anti-Tsal antibodies induced by various tsetse fly species in a range of hosts. We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species. In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts. In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a bottleneck.

No MeSH data available.


Related in: MedlinePlus

Detection of boosting in pigs after a period of non-exposure.Evolution of the anti-Tsal antibody responses in the competitive ELISA using TsalNb-5-HRP (A) and TsalNb-11-HRP (B) was evaluated in pigs (n = 2) originally exposed two-weekly for 6 weeks to the bites of 3 flies (= low exposure group) and re-exposed to the bites of 10 flies (indicated by an arrow) after a 2-month period of non-exposure. One non-exposed animal sampled during the priming experiment (Fig. 5) served as a negative control. The mean endpoint O.D.405nm of controls were 2.235 and 2.291 following detection with respectively TsalNb-5-HRP and TsalNb-11-HRP. Presented data are the percentages inhibition relative to the controls obtained for the plasmas of the individual pigs.
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pntd.0003456.g006: Detection of boosting in pigs after a period of non-exposure.Evolution of the anti-Tsal antibody responses in the competitive ELISA using TsalNb-5-HRP (A) and TsalNb-11-HRP (B) was evaluated in pigs (n = 2) originally exposed two-weekly for 6 weeks to the bites of 3 flies (= low exposure group) and re-exposed to the bites of 10 flies (indicated by an arrow) after a 2-month period of non-exposure. One non-exposed animal sampled during the priming experiment (Fig. 5) served as a negative control. The mean endpoint O.D.405nm of controls were 2.235 and 2.291 following detection with respectively TsalNb-5-HRP and TsalNb-11-HRP. Presented data are the percentages inhibition relative to the controls obtained for the plasmas of the individual pigs.

Mentions: Plasma samples were previously collected from pigs that were experimentally exposed to a low or a high tsetse fly bite regimen. Titration studies using plasma collected at the peak of anti-Tsal1 and anti-G. m. morsitans saliva antibody responses as determined earlier [18], indicated that best competition was obtained with undiluted porcine plasma (p < 0.05; S2 Fig.). Evaluation of the anti-Tsal responses over time with the competitive assay revealed similar kinetics as determined earlier with the G. m. morsitans saliva and rTsal1 based indirect ELISA. A steady decline in antibody titers was observed upon cessation of tsetse exposure (Fig. 5A&B). However, unlike the indirect ELISA, competitive ELISA test results did not correlate significantly with the intensity of tsetse exposure. Boosting of two immunized pigs from the low exposure group after a 2-month non-exposure period by the bites of 10 flies resulted in elevated anti-saliva antibody titers that were detectable in the competitive immunoassay (Fig. 6A&B). A good correlation between the TsalNb-5-HRP and TsalNb-11-HRP based tests was recorded with a Spearman correlation coefficient r of 0.95 (S3A Fig.). Area under the ROC curve (AUC = 0.99, S3B Fig.) for the TsalNb-5-HRP and TsalNb-11-HRP immunoassays were very high and exceeded AUCs that we reported previously for the porcine indirect ELISA using respectively G. m. morsitans saliva and rTsal1 as coating antigens (AUC = 0.96 and 0.83, [18]). Given the absence of a gold standard, we made a comparison of the new competitive test with the previously described indirect ELISA tests in terms of sensitivity, specificity, positive and negative predictive values (PPV and NPV) and accuracy determined for the available set of experimentally exposed porcine plasmas (nexposed= 90, nnon-exposed = 21, Table 2). These analyses showed that all assays under their specific test conditions had very good specificities (> 95%) and PPVs (> 98%). However, the competitive assay appeared to result in less false negative diagnoses, resulting in higher NPVs and hence increased sensitivity (> 95%) and accuracy (> 95%).


Description of a nanobody-based competitive immunoassay to detect tsetse fly exposure.

Caljon G, Hussain S, Vermeiren L, Van Den Abbeele J - PLoS Negl Trop Dis (2015)

Detection of boosting in pigs after a period of non-exposure.Evolution of the anti-Tsal antibody responses in the competitive ELISA using TsalNb-5-HRP (A) and TsalNb-11-HRP (B) was evaluated in pigs (n = 2) originally exposed two-weekly for 6 weeks to the bites of 3 flies (= low exposure group) and re-exposed to the bites of 10 flies (indicated by an arrow) after a 2-month period of non-exposure. One non-exposed animal sampled during the priming experiment (Fig. 5) served as a negative control. The mean endpoint O.D.405nm of controls were 2.235 and 2.291 following detection with respectively TsalNb-5-HRP and TsalNb-11-HRP. Presented data are the percentages inhibition relative to the controls obtained for the plasmas of the individual pigs.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4320081&req=5

pntd.0003456.g006: Detection of boosting in pigs after a period of non-exposure.Evolution of the anti-Tsal antibody responses in the competitive ELISA using TsalNb-5-HRP (A) and TsalNb-11-HRP (B) was evaluated in pigs (n = 2) originally exposed two-weekly for 6 weeks to the bites of 3 flies (= low exposure group) and re-exposed to the bites of 10 flies (indicated by an arrow) after a 2-month period of non-exposure. One non-exposed animal sampled during the priming experiment (Fig. 5) served as a negative control. The mean endpoint O.D.405nm of controls were 2.235 and 2.291 following detection with respectively TsalNb-5-HRP and TsalNb-11-HRP. Presented data are the percentages inhibition relative to the controls obtained for the plasmas of the individual pigs.
Mentions: Plasma samples were previously collected from pigs that were experimentally exposed to a low or a high tsetse fly bite regimen. Titration studies using plasma collected at the peak of anti-Tsal1 and anti-G. m. morsitans saliva antibody responses as determined earlier [18], indicated that best competition was obtained with undiluted porcine plasma (p < 0.05; S2 Fig.). Evaluation of the anti-Tsal responses over time with the competitive assay revealed similar kinetics as determined earlier with the G. m. morsitans saliva and rTsal1 based indirect ELISA. A steady decline in antibody titers was observed upon cessation of tsetse exposure (Fig. 5A&B). However, unlike the indirect ELISA, competitive ELISA test results did not correlate significantly with the intensity of tsetse exposure. Boosting of two immunized pigs from the low exposure group after a 2-month non-exposure period by the bites of 10 flies resulted in elevated anti-saliva antibody titers that were detectable in the competitive immunoassay (Fig. 6A&B). A good correlation between the TsalNb-5-HRP and TsalNb-11-HRP based tests was recorded with a Spearman correlation coefficient r of 0.95 (S3A Fig.). Area under the ROC curve (AUC = 0.99, S3B Fig.) for the TsalNb-5-HRP and TsalNb-11-HRP immunoassays were very high and exceeded AUCs that we reported previously for the porcine indirect ELISA using respectively G. m. morsitans saliva and rTsal1 as coating antigens (AUC = 0.96 and 0.83, [18]). Given the absence of a gold standard, we made a comparison of the new competitive test with the previously described indirect ELISA tests in terms of sensitivity, specificity, positive and negative predictive values (PPV and NPV) and accuracy determined for the available set of experimentally exposed porcine plasmas (nexposed= 90, nnon-exposed = 21, Table 2). These analyses showed that all assays under their specific test conditions had very good specificities (> 95%) and PPVs (> 98%). However, the competitive assay appeared to result in less false negative diagnoses, resulting in higher NPVs and hence increased sensitivity (> 95%) and accuracy (> 95%).

Bottom Line: We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species.In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts.In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a bottleneck.

View Article: PubMed Central - PubMed

Affiliation: Unit of Veterinary Protozoology, Department of Biomedical Sciences, Institute of Tropical Medicine Antwerp (ITM), Antwerp, Belgium; Unit of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium; Laboratory of Myeloid Cell Immunology, VIB, Brussels, Belgium.

ABSTRACT

Background: Tsetse flies are the main vectors of human and animal African trypanosomes. The Tsal proteins in tsetse fly saliva were previously identified as suitable biomarkers of bite exposure. A new competitive assay was conceived based on nanobody (Nb) technology to ameliorate the detection of anti-Tsal antibodies in mammalian hosts.

Methodology/principal findings: A camelid-derived Nb library was generated against the Glossina morsitans morsitans sialome and exploited to select Tsal specific Nbs. One of the three identified Nb families (family III, TsalNb-05 and TsalNb-11) was found suitable for anti-Tsal antibody detection in a competitive ELISA format. The competitive ELISA was able to detect exposure to a broad range of tsetse species (G. morsitans morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes) and did not cross-react with the other hematophagous insects (Stomoxys calcitrans and Tabanus yao). Using a collection of plasmas from tsetse-exposed pigs, the new test characteristics were compared with those of the previously described G. m. moristans and rTsal1 indirect ELISAs, revealing equally good specificities (> 95%) and positive predictive values (> 98%) but higher negative predictive values and hence increased sensitivity (> 95%) and accuracy (> 95%).

Conclusion/significance: We have developed a highly accurate Nb-based competitive immunoassay to detect specific anti-Tsal antibodies induced by various tsetse fly species in a range of hosts. We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species. In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts. In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a bottleneck.

No MeSH data available.


Related in: MedlinePlus