Gel filtration of purified rISG65 on a Superdex 200 10/300 GL column (GE Healthcare Lifesciences) in sodium phosphate buffer, pH7.5, O.5 M NaCl.

<p>A. Molecular weight markers (Sigma-Aldrich MWGF100) 1 = thyroglobulin 669 kDa, 2 = apoferritin 443 kDa, 3 = β-amylase 200 kDa, 4 = alcohol dehydrogenase 150 kDa, 5 = bovine serum albumin 66 kDa, 6 = carbonic anhydrase 29 kDa. B. Elution profile of rISG following one round of metal affinity chromatography purification.</p

Analysis of the deglycosylation of rISG65 using PNGase F (1.5 U) (Sigma-Aldrich, P7367) for two hours at 37°C.

<p>Coomassie blue stained pre-cast 4–12% BisTris gradient SDS-PAGE gel (Novex) using the MOPS running system; Precision Plus Protein Unstained Standards, BioRad (M). Lane 1: rISG65 (1 ug) before treatment with PNGase F, Lane 2: rISG65 (1 ug) after treatment with PNGase F.</p

SDS-PAGE analysis of the expression of rISG65, rLiTat1.3 and rLiTat1.5 by <i>Leishmania tarentolae</i> after purification following a single round of metal affinity chromatography.

<p>Coomassie stained 10% SDS-PAGE; Precision Plus Protein Unstained Standards, BioRad (M). Lane 1: rISG65, Lane 2: rLiTat1.3, Lane 3: rLiTat1.5. A = 1 μg and B = 10 μg protein.</p

Area under the curve (AUC) and its 95% confidence interval (CI), Youden index, percent sensitivity (Se %), percent specificity (Sp %) and their respective 95% CI recorded for the different antigens when tested with sera from 172 <i>g-</i>HAT patients, 119 non-<i>g-</i> HAT controls and 50 non-<i>r-</i>HAT controls.

<p>Area under the curve (AUC) and its 95% confidence interval (CI), Youden index, percent sensitivity (Se %), percent specificity (Sp %) and their respective 95% CI recorded for the different antigens when tested with sera from 172 <i>g-</i>HAT patients, 119 non-<i>g-</i> HAT controls and 50 non-<i>r-</i>HAT controls.</p

Receiver operator characteristic (ROC) curves and area under the curve (AUC) constructed from ELISA results obtained by testing sera from 172 <i>g-</i>HAT patients, 119 non-<i>g-</i>HAT controls and 50 non-<i>r-</i>HAT controls with nLiTat 1.3 (2 μg/ml), rLiTat 1.3 (4 μg/ml), nLiTat 1.5 (2 μg/ml), rLiTat 1.5 (4 μg/ml) and rISG65 (4 μg/ml).

<p>Receiver operator characteristic (ROC) curves and area under the curve (AUC) constructed from ELISA results obtained by testing sera from 172 <i>g-</i>HAT patients, 119 non-<i>g-</i>HAT controls and 50 non-<i>r-</i>HAT controls with nLiTat 1.3 (2 μg/ml), rLiTat 1.3 (4 μg/ml), nLiTat 1.5 (2 μg/ml), rLiTat 1.5 (4 μg/ml) and rISG65 (4 μg/ml).</p

Serum collection used in this study.

<p>Serum collection used in this study.</p

Pairwise difference and <i>p</i> value of the Chi square between the areas under the curve recorded for the different antigens when tested with sera from 172 <i>T</i>.<i>b</i>. <i>g-</i>HAT patients, 119 non-<i>g-</i>HAT controls and 50 non-<i>r-</i>HAT controls.

<p>* = significantly different</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

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

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