Fig 1.
Schematic illustration of the Nb library generation, panning and screening strategy.
The Nb library was generated from an alpaca that was immunized with T. congolense TC13 soluble proteome (s.p.). Parallel panning was performed on s.p. of T. congolense homologous strain (TC13) or heterologous strains (MF3cl2, STIB68 and TRT17). Then panning round 2 and 3 were screened on heterologous strains for colonies expressing anti-T. congolense s.p. Nbs. All the positive colonies were re-screened on TC13 s.p. as well as mouse serum in order to identify and eliminate those expressing non-specific Nbs.
Fig 2.
Specific recognition of T. congolense by the Nb474-ELISA.
(A) ELISA binding assay performed on trypanosome soluble proteome (s.p.) reveals specific recognition of T. congolense. (B) ELISA binding assay performed on sera collected from naive as well as T. congolense infected mice shows the ability of the ELISA to detect infected animals. (C) Assessment of the ELISA on pooled sera collected from mice infected with different species of trypanosomes indicates specific detection and cross-reaction with various strains of T. congolense. The OD450nm shown on the graph represent the average value recorded from the duplicate wells. Results are expressed +/- standard deviation (SD) and statistical analysis was performed by comparing the OD of non-infected animals with those of infected animals. n.s. = non-significant, ** p<0.01, *** p<0.001, **** p<0.0001.
Fig 3.
Effect of Berenil treatment intervention on antigenemia in mice.
(A) The progression of antigenemia and parasitemia with no treatment intervention. Antigen levels constantly remained above baseline (naive state) when there is active infection. (B) The progression of antigenemia and parasitemia before and after Berenil treatment. Berenil treatment cleared circulating parasites causing a sharp decline in the levels of circulating antigen. The OD450nm shown on the graph represent the average value recorded from pooled sera analyzed in duplicate. Results are representative of two experiments consisting of 5 mice per group and expressed as +/- standard deviation (SD). Arrows are showing time points where mice were infected with trypanosomes and treated with Berenil.
Fig 4.
Nb474-ELISA detected T. congolense infections in experimentally and naturally infected cattle.
(A) Examination of sera collected from naive and cattle experimentally infected with T. congolense IL1180. Average OD450nm recorded on sera of naive cattle was significantly lower than those of infected cattle. (B) Examination of a panel of field blood samples collected from various domestic animals positive for different endemic tropical parasites has shown that the ELISA efficiently detected samples in two field samples collected from cattle that were naturally infected with T. congolense. The OD450nm values plotted on the graph represents the average value recorded from the duplicate wells. ** p<0.01.
Table 1.
Performance of the Nb474-based ELISA on T. congolense IL1180 experimentally infected cattle and naive animals.
Fig 5.
Immunofluorescence and ELISA revealed that Nb474 targets a protein in the T. congolense BSF.
(A) Preliminary analysis of Alexa-labeled Nb474 binding to fixed and permeabilized T. congolense BSF parasites reveals green intra-cytoplasmic structures green (panels 3 and 4). (B) Co-localization of Alexa labeled Nb474 and anti-T. brucei aldolase MAb (Anti-TbALD) performed on T. congolense BSF shows the binding of the Nb matched with the Anti-TbALD. (C) The Nb474-ELISA specifically recognizes T. congolense BSF soluble proteome (s.p.), which is an indication of target uniqueness to the BSF.
Fig 6.
Detection of Nb474H immuno-affinity captured glycosomal aldolase.
(A) Nb474H immuno-affinity captured glycosomal aldolase (TcoALD) detected by SDS-PAGE under reducing conditions. Panel (top left): Native TcoALD was captured from the soluble proteome (s.p.) or infected sera on a 96-well ELISA coated with Nb474H, eluted from the plate and analysed on a 10% SDS-PAGE developed with silver staining. Lane M, protein ladder; lane 1, pure Nb474H; lane 2, pure Nb474B; lane 3, eluted protein captured from s.p. (6 μg); lane 4, eluted protein captured from infected sera (8 μg). Panel (top right): Native TcoALD was captured from secretome on nickel beads linked to Nb474H, eluted and analysed on a 10% SDS-PAGE developed with coomassie blue. Lane M, protein ladder; lane 1, Nb474H; lane 2, flow through; lane 3, wash; lane 4, eluted protein. Native TcoALD protein migrated at ±40 kDa (arrows, top) and the Nb474 migrated at ±15kDa (arrows, bottom). (B) Nb474H immuno-affinity captured TcoALD from T. congolense TC13 s.p. detected by Anti-T. brucei aldolase MAb (Anti-TbALD MAb) in ELISA. Bar (left): OD450nm levels in wells filled with coating buffer followed by addition of T. congolense TC13 s.p. and then Anti-TbALD MAb; bar (middle): OD450nm in wells coated with the Nb followed by addition of Anti-TbALD MAb; and bar (right): OD450nm in wells coated with the Nb followed by addition of T. congolense TC13 s.p. and then Anti-TbALD MAb. In the last step goat anti-mouse IgG conjugated to Horse radish peroxidase (HRP) was added to all the wells and then developed with 1-Step ultra 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. The OD450nm shown on the graph represent the average value of duplicate wells. *** p<0.001.
Fig 7.
ELISA binding assay performed on recombinant glycosomal aldolase expressed in E. coli.
Crude lysate prepared from cultures obtained before induction of protein expression (0 h) and after induction (18 h) were probed. (A) Anti-His IgG or (B) Anti-T. brucei aldolase MAb (Anti-TbALD MAb) was used for probing the presence of expressed aldolase in crude lysate coated on ELISA plate. Anti-His IgG detected all the three recombinant aldolase and Anti-TbALD MAb only detected trypanosomal aldolase (C) Nb474-based homologous sandwich ELISA (C) or (D) Nb474H-Anti-TbALD MAb heterologous sandwich ELISA was used to probe the presence of expressed aldolase in crude lysate. Both homologous (Nb474H-Nb474B) and heterologous (Nb474H-Anti-TbALD MAb) sandwich ELISA detected T. congolense aldolase only. The OD450nm shown on the graphs represent the average value recorded from the duplicate wells.