Nanobody-based Immunoassay for Trypanosoma congolense targeting Glycosomal Fructose 1,6-Bisphosphate Aldolase

Student thesis: Doctoral Thesis

Abstract

Yearly, infectious diseases cause untold mortalities and economic losses worldwide. While the existing curative measures could alleviate the situation, the inadequacies of diagnostics prevent early case detection. Technological advancement brought diagnostics that have high performance but they are laboratory-based, require skilled personnel, time consuming and costly. Altogether, they are inaccessible by rural dwellers where infectious diseases are highly prevalent. Delayed diagnosis causes mild infections to progress resulting in death, disability and transmissions. Moreover, the absence of effective tests prevents early detection of drug resistant parasites or persistent infections ascribed to counterfeit drugs.
Technology for transforming complex immunodiagnostics into point-of-care (POC) tests is well established. These POC tests detect antigen through monoclonal antibodies (MAbs) that are immobilized on a solid support or infection-induced host antibodies through immobilized antigen. Antigen detection reliably reveals ongoing infection unlike antibody detection whose specificities are compromised by persistence of antibodies even after clearance of infections. While antigen detecting POC tests would suffice in rural settings, efforts to develop them are thwarted by inability of the tests’ MAbs to outcompete infection-induced host antibodies which are directed against the same epitopes on the antigen thereby affecting sensitivity. The recombinant variable portion of a camelid heavy-chain antibody (cf. Nanobody) is able to overcome this challenge. Indeed, Nanobodies (Nbs) have a pronounced CDR3 thereby reaching cryptic epitopes inaccessible by short CDRs of the conventional antibodies and evading the competition.
This study explored the development of Nb-based antigen detection assay for African Animal Trypanosomiasis (AAT), which affects livestock industry in rural Sub-Saharan Africa. As a preliminary, a pilot surveillance study was conducted in a typical AAT endemic region (Uganda) and, indeed, trypanosome parasites were detected among other hemoparasites. T. congolense being the most predominant pathogenic trypanosome was chosen as a model parasite for the development of a Nb-based antigen detection Enzyme-linked Immunosorbent Assay (ELISA) that will be translated into a POC test device. The target biomarker of this immunoassay was the fructose 1,6-bisphosphate aldolase (aldolase) enzyme which is found in the glycosomes of trypanosomes. Because there is no gold standard for evaluation of the final assay, comparative performance of the nested ITS PCR and 18S-PCR-RFLP pan-trypanosome assays was performed for this purpose. Given that the nested ITS PCR could detect mixed infections better than the 18S-PCR-RFLP, this makes the test more reliable. Thus, once the Nb-based ELISA is translated into a future POC test, its performance should be evaluated against the nested ITS PCR.
Date of Award23 Jun 2016
Original languageEnglish
Awarding Institution
  • Vrije Universiteit Brussel

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