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

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

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

Average percent remaining activity of the mAb binding to the biotinylated synthetic peptides.

<p>The ability of human serum antibodies to bind to the mimotopic peptides selected with anti-VSG monoclonal antibodies was assessed by means of an inhibition ELISA. *: <i>p</i><0.05 and **: <i>p</i><0.01. Compared to the ten HAT negative sera, the nine HAT positive sera significantly inhibited the binding of anti-LiTat 1.5 mAb H12H3 to peptide 21, 22, 23, 28, C59 and C60 and the binding of anti-LiTat 1.3 mAb H13F7 to peptides 25, 60 and 61.</p

Amino acid sequences of peptides obtained by phage display with anti- VSG LiTat 1.5 mAb H12H3.

<p>The first sequence displayed is amino acid 265 to 288 of the protein sequence of VSG LiTat 1.5. Homologous amino acids are indicated in grey and amino acids that are identical to those of the VSG protein sequence are in bold and grey. All peptide sequences include the GGGS-spacer (connecting phage protein pIII and the random peptide in the original library) at the C-terminal. When expressed on the phage, the first cysteine of the C7C-peptides is preceded by alanine. AAA: eluted via antigen competition. PPP: eluted via acidification. Freq: number of times this sequence was found amongst seventy-nine selected phage clones. Name: name of the synthesised peptide.*: peptide selected for biotinylation. NW: not withheld. % identity: percentage identity of the peptide sequence with a stretch of 14 AA (AA 268 to 281) within the protein sequence of VSG LiTat 1.5. % RA mAb-VSG: percentage remaining of the mAb binding to its corresponding native VSG after inhibition by the synthetic peptide at 67 µg/ml.</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

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

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