An Anti-proteome Nanobody Library Approach Yields a Specific Immunoassay for Trypanosoma congolense Diagnosis Targeting Glycosomal Aldolase

Background Infectious diseases pose a severe worldwide threat to human and livestock health. While early diagnosis could enable prompt preventive interventions, the majority of diseases are found in rural settings where basic laboratory facilities are scarce. Under such field conditions, point-of-care immunoassays provide an appropriate solution for rapid and reliable diagnosis. The limiting steps in the development of the assay are the identification of a suitable target antigen and the selection of appropriate high affinity capture and detection antibodies. To meet these challenges, we describe the development of a Nanobody (Nb)-based antigen detection assay generated from a Nb library directed against the soluble proteome of an infectious agent. In this study, Trypanosoma congolense was chosen as a model system. Methodology/Principal Findings An alpaca was vaccinated with whole-parasite soluble proteome to generate a Nb library from which the most potent T. congolense specific Nb sandwich immunoassay (Nb474H-Nb474B) was selected. First, the Nb474-homologous sandwich ELISA (Nb474-ELISA) was shown to detect experimental infections with high Positive Predictive Value (98%), Sensitivity (87%) and Specificity (94%). Second, it was demonstrated under experimental conditions that the assay serves as test-of-cure after Berenil treatment. Finally, this assay allowed target antigen identification. The latter was independently purified through immuno-capturing from (i) T. congolense soluble proteome, (ii) T. congolense secretome preparation and (iii) sera of T. congolense infected mice. Subsequent mass spectrometry analysis identified the target as T. congolense glycosomal aldolase. Conclusions/Significance The results show that glycosomal aldolase is a candidate biomarker for active T. congolense infections. In addition, and by proof-of-principle, the data demonstrate that the Nb strategy devised here offers a unique approach to both diagnostic development and target discovery that could be widely applied to other infectious diseases.
Author Summary The lack of diagnostic tests that are sensitive, affordable and user-friendly is the impediment to early detection and containment of infectious diseases. For these reasons, African trypanosomosis continues to pose serious threat to the communities that are unable to access laboratory services. Assays that are able to address above-mentioned challenges would greatly reduce the disease burden. Although few relatively sensitive agglutination assays for some forms of African trypanosomosis have been adapted to field conditions, the tests are not reliable indicators of active infections given the fact that they only detect antibodies. The barrier in the development of alternative tests capable of revealing ongoing infections through antigen detection is the lack of potent monoclonal antibodies that can compete with infection-induced host antibodies for the circulating parasite antigens. Using T. congolense as a model system, we demonstrate that Nanobodies (Nbs) targeting the parasite glycosomal aldolase can detect active infections. The strategy described addresses the technical shortcoming of conventional monoclonal antibody-based assay development by adopting an unbiased proteome screening approach combined with a phage panning strategy that is adapted to avoid interference of the infection-induced host antibody response. Hence, this study shows prospect for future development of Nb-based tests for other infectious diseases.