Theoretical and experimental epitope mapping of thymosin β4

Becker, Stefan; Armbruster, Franz Paul; Muller, B.; Echner, Hartmut; Kapurnotu, A.; Livaniou, Evangelia; Mihelić, M.; Stoeva, Stanka; Voelter, Wolfgang

doi:10.1016/0022-1759(94)90150-3

Cited by 6 publications

(6 citation statements)

References 10 publications

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“…Linear epitopes are then defined by identifying the peptides that are most strongly associated with antibodies developed against the full-sized antigen. This methodology has been used successfully in numerous cases [16-19]. …”

Section: Introductionmentioning

confidence: 99%

Chagas disease-specific antigens: characterization of epitopes in CRA/FRA by synthetic peptide mapping and evaluation by ELISA-peptide assay

et al. 2013

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BackgroundThe identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines. In this study, epitopes in the cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins from Trypanosoma cruzi were identified using synthetic peptide techniques and pooled sera from Chagasic patients. The epitopes were further assayed with an ELISA assay based on synthetic peptides.MethodsTwenty-two overlapping synthetic peptides representing the coding sequence of the T. cruzi CRA and FRA proteins were assessed by a Spot-synthesis array analysis using sera donated by patients with Chagas disease. Shorter peptides were selected that represented the determined epitopes and synthesized by solid phase synthesis to evaluate the patterns of cross-reactivities and discrimination through an ELISA-diagnostic assay.ResultsThe peptide Spot-synthesis array successfully identified two IgG antigenic determinants in the CRA protein and four in FRA. Bioinformatics suggested that the CRA antigens were unique to T. cruzi while the FRA antigen showed similarity with sequences present within various proteins from Leishmania sp. Subsequently, shorter peptides representing the CRA-1, CRA-2 and FRA-1 epitopes were synthesized by solid phase synthesis and assayed by an ELISA-diagnostic assay. The CRA antigens gave a high discrimination between Chagasic, Leishmaniasis and T. cruzi-uninfected serum. A sensitivity and specificity of 100% was calculated for CRA. While the FRA antigen showed a slightly lower sensitivity (91.6%), its specificity was only 60%.ConclusionsThe epitopes recognized by human anti-T. cruzi antibodies have been precisely located in two biomarkers of T. cruzi, CRA and FRA. The results from screening a panel of patient sera through an ELISA assay based on peptides representing these epitopes strongly suggest that the sequences from CRA would be useful for the development of diagnostic reagents that could improve upon the sensitivity and specificity of currently available diagnostic tests. Overall, the results provide further evidence of the usefulness of identifying specific linear B-cell epitopes for improving diagnostic tools.

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Section: Introductionmentioning

confidence: 99%

Chagas disease-specific antigens: characterization of epitopes in CRA/FRA by synthetic peptide mapping and evaluation by ELISA-peptide assay

et al. 2013

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“…HTC cells were grown in RPMI 1640 medium containing 10% fetal bovine serum for 12 h at a concentration of 2 × 10 6 cells/mL/well in 24‐well cell culture plates. Duplicate cultures were then treated with either phosphate‐buffered saline (PBS) (control) or 1 μg LPS or 10 μg PG‐PS in PBS for an additional 12 h. At the end of incubation, the cells were centrifuged at 250 g and the supernatant was used to determine Tβ‐4 concentration by a competitive enzyme immunoassay using a rabbit anti‐human Tβ‐4 antiserum (Abcam, Cambridge, MA) and human Tβ‐4 (ProSpec‐Tany TechnoGene Ltd., Rehovot, Israel) as the coating antigen and the standard 6, 7 . The nitrite and the interleukin‐6 (IL‐6) concentrations in the culture supernatant was determined using Griess reagent 8 and a B9 hybridoma bioassay 9 as described earlier 3, 4 …”

Section: Methodsmentioning

confidence: 99%

“…Duplicate cultures were then treated with either phosphate-buffered saline (PBS) (control) or 1 g LPS or 10 g PG-PS in PBS for an additional 12 h. At the end of incubation, the cells were centrifuged at 250 g and the supernatant was used to determine T␤-4 concentration by a competitive enzyme immunoassay using a rabbit anti-human T␤-4 antiserum (Abcam, Cambridge, MA) and human T␤-4 (ProSpec-Tany TechnoGene Ltd., Rehovot, Israel) as the coating antigen and the standard. 6,7 The nitrite and the interleukin-6 (IL-6) concentrations in the culture supernatant was determined using Griess reagent 8 and a B9 hybridoma bioassay 9 as described earlier. 3,4 After removing the medium, the cells were washed once with sterile PBS and DNA-free total cellular RNA was purified using RNAeasy and On-column DNA digestion kits (Qiagen Corp, Chatsworth, CA).…”

Section: Effects Of Immunomodulators On T␤-4 Gene Expressionmentioning

confidence: 99%

Identification and Characterization of Thymosin β‐4 in Chicken Macrophages Using Whole Cell MALDI‐TOF

Kannan¹,

Rath²,

Liyanage³

et al. 2007

Annals of the New York Academy of Sciences

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The aim of the study was to determine chicken monocyte- and granulocyte-associated peptides and proteins using "whole cell" matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and to characterize the peptides based on their abundance. The mass spectra showed a prominent peak at m/z 4963 in monocytes/macrophages but not in the granulocytes. Subsequent purification and characterization of the m/z 4963 peptide from an avian macrophage cell line HTC, revealed it to be thymosin beta-4 (Tbeta-4), an actin-modulating peptide. HTC cells when treated with bacterial lipopolysaccharide and peptidoglycan to determine the modulation of Tbeta-4 gene expression or its secretion, showed no changes.

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“…According to the results received from the antigenic index plots (Figures 1, 2), the troponin-T segment 34 -62 should be indeed a peptide fragment, suitable for the development of specific antibodies, recognizing the selected segment as well as the parent protein, as we could demonstrate in the past in connection with the development of specific immunoassays for quantitative determinations of thymosin β 9 [36], thymosin β 4 [37,38], transmembrane glycoprotein gp41 env [39], human relaxin [40] or immunostaining experiments of β-thymosins [41,25]. In our laboratory, solution [42], as well as solid phase [43 -45, 26] peptide syntheses are well established for many years, and improved, highly efficient strategies are available.…”

Section: Synthesis Of Troponin-t Fragment 34 -62mentioning

confidence: 98%

Analytical Tools for Rapid, Sensitive, Quantitative Identification of Potential Meat Quality Markers

Voelter¹,

Stoeva

Echner

et al. 2000

J. prakt. Chem.

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Conversion of muscle tissue to meat, including tenderization, is a complex process. As the consumer considers meat tenderness to be the most important criterion for meat quality, this conversion should be elucidated on a biomolecular basis. With such a biochemical background, specific sensitive analytical tools could be developed which would correlate physicochemical parameters with the degree of tenderness.Various events take place at the level of the myofibrils which have been correlated with meat tenderness. These include Z-disk weakening [1], degradation of desmin or titin, leading to fragmentation or weakening of myofibrils, the disappearance of troponin-T and simultaneous appearance of 28 -32 kDa proteins or detection of a 110 kDa protein fragment, as monitored thoughout the conditioning period by electron microscopic studies and SDS polyacrylamide gel electroAbstract. Myofibrillar extracts from bovine Musculus longissimus dorsi (MLD) were subjected to SDS-PAGE, electroblotted and fragments of the 30 kDa band determined by internal and N-terminal Edman sequencing, giving unequivocal proof, troponin-T (TNT) to be the origin of this band. Based on the N-terminal primary sequence of the 30 kDa band, a peptide with high antigenic sites was synthesized, conjugated to keyhole limpet hemocyanin (KLH), antibodies were generated and an enzyme-linked immunosorbent assay (ELI-SA) was developed for the determination of TNT concentrations in meat samples. For routine determinations of meat quality markers it seems more convenient to analyse soluble meat extracts, produced by trichloroacetic acid (TCA) or HCl treatment. In the supernatants of TCA-treated MLDs, prominent peptide fragments from glyceraldehyde-3-phosphate Keywords: Capillary electrophoresis, Immunoassays, Peptides, Proteins, Meat quality markers phoresis, respectively [2 -7]. In particular, the disappearance of troponin-T and the concomitant appearance of 28 -32 kDa proteins seem to be good indicators of post-mortem proteolysis. However, the origin of the 30 kDa fragments was speculative and not determined on a structural basis [8 -10]. Therefore, we recently applied the technique of internal microsequencing to provide for the first time unequivocal proof for the origin of the 30 kDa protein [11]. The 30 kDa protein transferred to ProBlott and Immobilon membranes was digested with trypsin, the peptide mixture separated with HPLC, selected peptides isolated and sequenced. Eight peptides, varying from 5 to 13 amino acid residues, displayed between 50 and 100% homology to rabbit troponin-T, according to our protein homology search. As SDS-PAGE operations are tedious, inconvenient and time-consuming, we suggest in this communication could be separated by HPLC and identified by Edman degradation. Both fragments were found to increase with ageing and might become useful indicators of meat quality. After HPLC separation and structure elucidation of MLD HCl extracts, fructose-biphosphate aldolase, creatine kinase, glyceraldehyde-3-phosphate dehydrogenase and myogl...

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Theoretical and experimental epitope mapping of thymosin β4

Cited by 6 publications

References 10 publications

Chagas disease-specific antigens: characterization of epitopes in CRA/FRA by synthetic peptide mapping and evaluation by ELISA-peptide assay

Chagas disease-specific antigens: characterization of epitopes in CRA/FRA by synthetic peptide mapping and evaluation by ELISA-peptide assay

Identification and Characterization of Thymosin β‐4 in Chicken Macrophages Using Whole Cell MALDI‐TOF

Analytical Tools for Rapid, Sensitive, Quantitative Identification of Potential Meat Quality Markers

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