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

Abstract

BackgroundInfectious 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 FindingsAn 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/SignificanceThe 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.

Transcriptional Profiling of Midguts Prepared from Trypanosoma/T. congolense-Positive Glossina palpalis palpalis Collected from Two Distinct Cameroonian Foci: Coordinated Signatures of the Midguts’ Remodeling As T. congolense-Supportive Niches

“…Another similarity between the parasites is that their mantle, which consists of a variant surface glycoprotein, allows them to evade the host’s immune system by means of antigenic variations ( 19 – 21 ), thus rendering ineffective any vaccine approaches to fight HAT or AAT. Nevertheless, progress has been made in rapid diagnosis ( 22 ) and therapy that uses a nifurtimox–elfornitine combination in the treatment of the second phase of HAT ( 23 ). Furthermore, besides the use of trypanocidal drugs, the incidence of AAT can be lowered by introducing trypanotolerant cattle into AAT-infected area or through the antibody-mediated inhibition of trypanosome-secreted proteins involved in the parasite pathogenic process ( 24 , 25 ).…”

Use of camel single-domain antibodies for the diagnosis and treatment of zoonotic diseases

“…A VHH (Nb474) directed against T. congolese aldolase (TcoALD) has been developped for a sandwich immunoassay. This VHH is highly specific and did not recognize other trypanosomes such as T. brucei brucei, T. vivax and T. evansi [65,66].…”

Paradigm shift in the diagnosis of peste des petits ruminants: scoping review

“…Therefore, the use of nanobodies may circumvent binding interference caused by the host's antibody response. Thus, nanobodies should detect both free antigens as well as those bound by host antibodies, which would make nanobodies based diagnostic tests rather attractive [138]. In contrast, the large size of MAbs prevents them from reaching cryptic epitopes andhost IgG molecules might as well conceal the epitopes from the MAbs employed in the diagnostic test [139].…”