Abstract
BackgroundTsetse 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 FindingsA 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/SignificanceWe 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.
Highlights
Control of the tsetse fly vector population represents an important component of the fight against Human and Animal African Trypanosomiasis (HAT and AAT)
Our previous studies have revealed that the saliva of the savannah tsetse fly (Glossina morsitans morsitans) and the main constituting tsetse salivary gland (Tsal) proteins are sensitive immunological probes to detect contact with tsetse flies
A nanobody (Nb) library was generated against tsetse salivary gland proteins and used to select Nbs against the highly immunogenic Tsal proteins by a procedure of phage display and selection for binding onto the recombinant Tsal proteins
Summary
Control of the tsetse fly vector population represents an important component of the fight against Human and Animal African Trypanosomiasis (HAT and AAT). Studies using various tsetse fly species have shown that salivary components are immunogenic in mice, rabbits, cattle and humans with the induction of antibodies [7,8,9,10,11,12]. The 43–45 kDa tsetse salivary gland (Tsal) protein family, encoded by 3 tsal genes that colocalize to a 10-kb locus in the tsetse fly genome [19], was found to be highly immunogenic with immunoglobulin responses detected in humans living in Uganda [7], Democratic Republic of Congo [10] and Guinea [8]. Further exploiting the highly immunogenic nature of the Tsal proteins, rTsal was evaluated as antigen in an indirect ELISA test and shown adequate to detect tsetse induced antibody responses in experimentally exposed pigs [18]. A new competitive assay was conceived based on nanobody (Nb) technology to ameliorate the detection of anti-Tsal antibodies in mammalian hosts
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