Homologous and heterologous sandwich ELISAs on the <i>Tco</i>ALD variants.

<p>(A.) Homologous sandwich ELISA on the <i>Tco</i>ALD variants in which His-tagged (Nb474H) and biotinylated (Nb474B) Nb474 were employed as capturing and detecting antibodies, respectively. (B.) Heterologous sandwich ELISA on the <i>Tco</i>ALD variants in which biotinylated Nb474 (Nb474B) and mouse anti-His IgG were employed as capturing and detecting antibodies, respectively.</p

Investigation of the stoichiometry of the Nb474-<i>Tco</i>ALD complex by analytical SEC.

<p>(A.-G.) Analytical SEC on the purified <i>Tco</i>ALD (A.), Nb474 (B.) and samples containing <i>Tco</i>ALD and Nb474 mixed at different molar ratios: 4:1 (C.), 4:2 (D.), 4:3 (E.), 4:4 (F.), and 4:6 (G.). All experiments were performed on a Superdex 200 HR 10/30 column. The black and grey traces represent the chromatograms of the different protein samples and the BioRAD gel filtration standard, respectively. In all figures, the inset shows an SDS-PAGE analysis of the elution peaks. <i>Tco</i>ALD (MM = 42.6 kDa) and Nb474 (MM = 15.8 kDa) are indicated by the orange and black arrows, respectively. <i>Lane M</i>, Prestained Protein Molecular Weight Marker (Fermentas). (H.) The calibration of the Superdex 200 HR 10/30 column that allows estimation of molecular mass based on the sample’s elution volume. The values between brackets indicate the estimated molecular mass versus the theoretical mass of the sample under investigation.</p

Data collection and refinement statistics.

<p>Statistics for the highest resolution shell are shown in parentheses.</p

Immunofluorescence and ELISA revealed that Nb474 targets a protein in the <i>T</i>. <i>congolense</i> BSF.

<p>(A) Preliminary analysis of Alexa-labeled Nb474 binding to fixed and permeabilized <i>T</i>. <i>congolense</i> BSF parasites reveals green intra-cytoplasmic structures green (panels 3 and 4). (B) Co-localization of Alexa labeled Nb474 and anti-<i>T</i>. <i>brucei</i> aldolase MAb (Anti-<i>Tb</i>ALD) performed on <i>T</i>. <i>congolense</i> BSF shows the binding of the Nb matched with the Anti-<i>Tb</i>ALD. (C) The Nb474-ELISA specifically recognizes <i>T</i>. <i>congolense</i> BSF soluble proteome (s.p.), which is an indication of target uniqueness to the BSF.</p

Kinetic parameters of the Nb474-<i>Tco</i>ALD^WT and Nb474-<i>Tco</i>ALD^A77E interactions.

<p>Kinetic parameters of the Nb474-<i>Tco</i>ALD^WT and Nb474-<i>Tco</i>ALD^A77E interactions.</p

Crystal structure of the (Nb474-<i>Tco</i>ALD)₄ complex.

<p>(A.) Overview of the entire complex. The <i>Tco</i>ALD tetramer and Nb474 are colored in orange and grey, respectively. The A and B dimer interfaces of <i>Tco</i>ALD are indicated, together with the distances between the Nb474 epitopes relative to these interfaces. (B.) Close-up of the interaction between Nb474 and a <i>Tco</i>ALD monomer. Color codes are as in (A.) and the Nb474 CDRs are indicated in different colors (CDR1, yellow; CDR2, green; CDR3, pink). (C.) Amino acid sequence of Nb474. The CDRs are highlighted in the same colors as in (B.). The residues marked by the asterisk ‘*’ are part of the Nb474 paratope and are involved in epitope binding. More details given in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005932#pntd.0005932.s003" target="_blank">S3 Fig</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005932#pntd.0005932.s006" target="_blank">S1 Table</a>.</p

Comparison of <i>Tco</i>ALD to other trypanosomatid aldolases.

<p>(A.) Sequence alignment between <i>Tco</i>ALD, <i>Tb</i>ALD, and <i>Lm</i>ALD. The colored bars above the sequence alignment represent the percentage of sequence identity: green (100%), green-brown (between 30% and 100%), and red (below 30%). The corresponding secondary structure elements are shown below the sequence alignment. Cylinders and arrows represent α-helices and β-sheets, respectively. The <i>Tco</i>ALD residues involved in the interaction with Nb474 are marked by an asterisk ‘*’. The residues highlighted in blue were selected for site-specific mutagenesis. (B.) Stereo view of a superposition of the crystal structures of the Nb474-<i>Tco</i>ALD complex (this work, color code as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005932#pntd.0005932.g002" target="_blank">Fig 2</a>), <i>Tb</i>ALD (PDB ID: 1F2J, [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005932#pntd.0005932.ref026" target="_blank">26</a>], depicted in cyan), and <i>Lm</i>ALD (PDB ID: 1EPX, [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005932#pntd.0005932.ref026" target="_blank">26</a>], depicted in blue). Those residues that are located in the vicinity of or on the <i>Tco</i>ALD epitope recognized by Nb474 and are distinct between the three trypanosomatid aldolases are shown in stick representation.</p

Effect of Berenil treatment intervention on antigenemia in mice.

<p>(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 OD_450nm 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.</p

Detection of Nb474H immuno-affinity captured glycosomal aldolase.

<p>(A) Nb474H immuno-affinity captured glycosomal aldolase (<i>Tco</i>ALD) detected by SDS-PAGE under reducing conditions. Panel (<i>top left</i>): Native <i>Tco</i>ALD 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 (<i>top right</i>): Native <i>Tco</i>ALD 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 <i>Tco</i>ALD protein migrated at ±40 kDa (arrows, top) and the Nb474 migrated at ±15kDa (arrows, bottom). (B) Nb474H immuno-affinity captured <i>Tco</i>ALD from <i>T</i>. <i>congolense</i> TC13 s.p. detected by Anti-<i>T</i>. <i>brucei</i> aldolase MAb (Anti-<i>Tb</i>ALD MAb) in ELISA. Bar (<i>left</i>): OD450nm levels in wells filled with coating buffer followed by addition of <i>T</i>. <i>congolense</i> TC13 s.p. and then Anti-TbALD MAb; bar (<i>middle</i>): OD450nm in wells coated with the Nb followed by addition of Anti-<i>Tb</i>ALD MAb; and bar (<i>right</i>): OD450nm in wells coated with the Nb followed by addition of <i>T</i>. <i>congolense</i> TC13 s.p. and then Anti-<i>Tb</i>ALD 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.</p

A heat map readily identifies the optimal practical set-up to conduct the Nb474-based homologous immunoassay.

<p>Using a checkerboard system, varying amounts of capturing and detecting Nb474 were employed to identify those conditions yielding the highest ELISA signal. The details of this approach are described in [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005932#pntd.0005932.ref016" target="_blank">16</a>].</p