Alignment on VSG LiTat 1.5 of peptides selected with anti-VSG LiTat 1.5 antibody fractions.

<p>Homologous sequences between phage displayed peptides and/or the protein sequence of VSG LiTat 1.5 are indicated in grey. Amino acids that are identical to those of the VSG protein sequence are in bold and grey. All peptide sequences include the GGGS-spacer at the C-terminus. Maximum % identity: percentage identity of the peptide sequence with a corresponding stretch of sixteen AA within the protein sequence of VSG LiTat 1.5. Synth peptide: name of the synthesised peptide.</p

Peptide sequences of phage clones selected with human anti-VSG LiTat 1.3 antibodies.

<p>3-3-H3(1) and 3-3-H3(2) are two different phage clones, na: not applicable, SD: standard deviation,</p>*<p>not withheld: similar to 3-2-G5 & 3-2-G10.</p><p>The phage clones were selected after two or three positive (pos) selections. The peptide sequences that were expressed by these phage clones are given in column three. The average OD_c in sandwich ELISA, using nine purified antibody fractions as capture antibody, is shown in column four. If the peptide sequence was synthesised, the name of the biotinylated peptide is given in column five.</p

Mapping of peptide 5-2-D3.

<p>Peptide 5-2-D3 could be mapped (orange) with AA E 168|N 164|D 152|G 153|T 150|K 146|L 144|A 141 on the three-dimensional model of a VSG LiTat 1.5 N-terminal domain monomer by means of 3DEX and Chimera.</p

Evaluation of the potential of the biotinylated peptides for diagnosis of <i>T.b. gambiense</i> HAT.

<p>The ability of biotinylated synthetic peptides to bind human serum antibodies in 102 HAT positive and 102 endemic negative control sera was assessed by indirect ELISA. The area under the receiver operator characteristics curve (AUC) and the sensitivity and specificity at maximum Youden index are shown with 95% confidence intervals (CI).</p

Peptide sequences of phage clones selected with human anti VSG LiTat 1.5 antibodies.

<p>na: not applicable, SD: standard deviation,</p>*<p>not withheld: similar to 5-3-C1.</p><p>The phage clones were selected after 1, 2 or 3 positive selections. The peptide sequences that were expressed by these phage clones are given in column three. The average OD_c in sandwich ELISA, using nine purified antibody fractions as capture antibody, is shown in column four. If the peptide sequence was synthesised, the name of the biotinylated peptide is given in column five.</p

Expression of rLiTat 1. 5_{H-SUMO-33-426-Strep} by <i>Pichia pastoris</i>.

<p>A. Western blot with anti-His tag antibody; B: Western blot with anti-Strep tag antibody; C: Coomassie stained 12% SDS-PAGE gel; Protein markers (M): ColorPlus Prestained Protein Marker (NEB; A and B) and LMW-SDS protein marker (GE Healthcare; C); lane 1: supernatant of transfected <i>Pichia pastoris</i> M5 after 25 h induction; lane 2: supernatant of transfected <i>Pichia pastoris</i> M5 after 44 h induction; lane 3: Ni-NTA purified recombinant LiTat 1.5; lane 4: 20× concentrated flow-through of Ni-NTA purified supernatant.</p

Expression of rLiTat 1. 3_{H-SUMO-24-372-Strep} by <i>Pichia pastoris</i>.

<p>A. Western blot with anti-His tag antibody; B: Western blot with anti-Strep tag antibody; C: Coomassie stained 12% SDS-PAGE gel; Protein markers (M): ColorPlus Prestained Protein Marker (NEB; A and B) and LMW-SDS protein marker (GE Healthcare; C); lane 1: supernatant of transfected <i>Pichia pastoris</i> M5 after 25 h induction; lane 2: supernatant of transfected <i>Pichia pastoris</i> M5 after 44 h induction; lane 3: Ni-NTA purified recombinant LiTat 1.3; lane 4: 20× concentrated flow-through of Ni-NTA purified supernatant.</p

Receiver operator characteristic (ROC) curves and area under the curve (AUC) constructed from ELISA results obtained with native LiTat 1.3 (2 µg/ml), rLiTat 1.3_{H-SUMO-24-372-Strep} (4 µg/ml), native LiTat 1.5 (2 µg/ml), rLiTat 1.5_{H-SUMO-33-426-Strep} (4 µg/ml) and mixtures of both native (1+1 µg/ml) and recombinant (2+2 µg/ml) antigens tested with sera from 88 <i>T.b. gambiense</i> HAT patients and 74 non-HAT controls.

<p>Receiver operator characteristic (ROC) curves and area under the curve (AUC) constructed from ELISA results obtained with native LiTat 1.3 (2 µg/ml), rLiTat 1.3_{H-SUMO-24-372-Strep} (4 µg/ml), native LiTat 1.5 (2 µg/ml), rLiTat 1.5_{H-SUMO-33-426-Strep} (4 µg/ml) and mixtures of both native (1+1 µg/ml) and recombinant (2+2 µg/ml) antigens tested with sera from 88 <i>T.b. gambiense</i> HAT patients and 74 non-HAT controls.</p

Percent positivity cut-off (max. Youden index) and sensitivity and specificity with 95% confidence intervals of the different antigens tested in ELISA with sera from 88 <i>T.b. gambiense</i> HAT patients and 74 non-HAT controls.

<p><b>*</b> significant difference between recombinant and native.</p

Box blots of the percent positivity (PP) obtained in ELISA with respectively native LiTat 1.3 (2 µg/ml), rLiTat 1.3_{H-SUMO-24-372-Strep} (4 µg/ml), native LiTat 1.5 (2 µg/ml), rLiTat 1.5_{H-SUMO-33-426-Strep} (4 µg/ml) and mixtures of both native (1+1 µg/ml) and recombinant (2+2 µg/ml) antigens tested with sera from 88 <i>T.b. gambiense</i> HAT patients and 74 non-HAT controls.

<p>Box blots of the percent positivity (PP) obtained in ELISA with respectively native LiTat 1.3 (2 µg/ml), rLiTat 1.3_{H-SUMO-24-372-Strep} (4 µg/ml), native LiTat 1.5 (2 µg/ml), rLiTat 1.5_{H-SUMO-33-426-Strep} (4 µg/ml) and mixtures of both native (1+1 µg/ml) and recombinant (2+2 µg/ml) antigens tested with sera from 88 <i>T.b. gambiense</i> HAT patients and 74 non-HAT controls.</p