16 research outputs found

    Identification of Peptide Mimotopes of Trypanosoma brucei gambiense Variant Surface Glycoproteins

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    The control of human African trypanosomiasis or sleeping sickness, a deadly disease in sub-Saharan Africa, mainly depends on a correct diagnosis and treatment. The aim of our study was to identify mimotopic peptides (mimotopes) that may replace the native proteins in antibody detection tests for sleeping sickness and hereby improve the diagnostic sensitivity and specificity. We selected peptide expressing phages from the PhD.-12 and PhD.-C7C phage display libraries with mouse monoclonal antibodies specific to variant surface glycoprotein (VSG) LiTat 1.3 or LiTat 1.5 of Trypanosoma brucei gambiense. The peptide coding genes of the selected phages were sequenced and the corresponding peptides were synthesised. Several of the synthetic peptides were confirmed as mimotopes for VSG LiTat 1.3 or LiTat 1.5 since they were able to inhibit the binding of their homologous monoclonal to the corresponding VSG. These peptides were biotinylated and their diagnostic potential was assessed with human sera. We successfully demonstrated that human sleeping sickness sera recognise some of the mimotopes of VSG LiTat 1.3 and LiTat 1.5, indicating the diagnostic potential of such peptides

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

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    <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.

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    <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<sub>c</sub> 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

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

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    <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.

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    <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<sub>c</sub> 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.

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    <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

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

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    <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 ”g/ml.</p
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