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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

Competitive inhibition of TsalNb binding by immune plasma.Representatives of the identified anti-Tsal Nb families (TsalNb-1, TsalNb-9 and TsalNb-5,8&11) were covalently conjugated to HRP and evaluated for Tsal-binding (O.D.405nm) following incubation of the coated G. m. morsitans saliva with tsetse exposed immune or naive rabbit plasma. Significance levels based on two-way analysis of variance are indicated in the graphs (*** p<0.001).
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pntd.0003456.g003: Competitive inhibition of TsalNb binding by immune plasma.Representatives of the identified anti-Tsal Nb families (TsalNb-1, TsalNb-9 and TsalNb-5,8&11) were covalently conjugated to HRP and evaluated for Tsal-binding (O.D.405nm) following incubation of the coated G. m. morsitans saliva with tsetse exposed immune or naive rabbit plasma. Significance levels based on two-way analysis of variance are indicated in the graphs (*** p<0.001).

Mentions: Representatives of the 3 Tsal Nb families (TsalNb-1, TsalNb-9 and TsalNb-5,8&11) were chemically conjugated to horseradish peroxidase (HRP), purified by size exclusion chromatography to remove unlabeled Nb and used as detection moieties in the G. m. morsitans saliva based ELISA assay. Evaluation of the inhibition of Nb binding by immune plasma from tsetse fly exposed rabbits, provided strong statistical support for a significant competition with TsalNb-5,8&11 (p < 0.001, Fig. 3), representatives of TsalNb family III. To confirm the potential of TsalNb family III for the development of a competitive anti-Tsal antibody detection assay, the two different Nbs with the highest naive/immune O.D. ratios—TsalNb-5 and TsalNb-11—were HRP conjugated in an independent labeling reaction and purified (S1A Fig.), titrated to determine the optimal working Nb-HRP dilution (S1B Fig.) and evaluated in a competitive assay using various rabbit serum concentrations (S1C Fig.). A strong inhibition of Nb binding by the immune serum was observed for the two Nb-HRP conjugates.


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)

Competitive inhibition of TsalNb binding by immune plasma.Representatives of the identified anti-Tsal Nb families (TsalNb-1, TsalNb-9 and TsalNb-5,8&11) were covalently conjugated to HRP and evaluated for Tsal-binding (O.D.405nm) following incubation of the coated G. m. morsitans saliva with tsetse exposed immune or naive rabbit plasma. Significance levels based on two-way analysis of variance are indicated in the graphs (*** p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003456.g003: Competitive inhibition of TsalNb binding by immune plasma.Representatives of the identified anti-Tsal Nb families (TsalNb-1, TsalNb-9 and TsalNb-5,8&11) were covalently conjugated to HRP and evaluated for Tsal-binding (O.D.405nm) following incubation of the coated G. m. morsitans saliva with tsetse exposed immune or naive rabbit plasma. Significance levels based on two-way analysis of variance are indicated in the graphs (*** p<0.001).
Mentions: Representatives of the 3 Tsal Nb families (TsalNb-1, TsalNb-9 and TsalNb-5,8&11) were chemically conjugated to horseradish peroxidase (HRP), purified by size exclusion chromatography to remove unlabeled Nb and used as detection moieties in the G. m. morsitans saliva based ELISA assay. Evaluation of the inhibition of Nb binding by immune plasma from tsetse fly exposed rabbits, provided strong statistical support for a significant competition with TsalNb-5,8&11 (p < 0.001, Fig. 3), representatives of TsalNb family III. To confirm the potential of TsalNb family III for the development of a competitive anti-Tsal antibody detection assay, the two different Nbs with the highest naive/immune O.D. ratios—TsalNb-5 and TsalNb-11—were HRP conjugated in an independent labeling reaction and purified (S1A Fig.), titrated to determine the optimal working Nb-HRP dilution (S1B Fig.) and evaluated in a competitive assay using various rabbit serum concentrations (S1C Fig.). A strong inhibition of Nb binding by the immune serum was observed for the two Nb-HRP conjugates.

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