A thymosin β4 ELISA using an antibody against the N terminal fragment thymosin β4[1–14]

Livaniou, Evangelia; Mihelić, M.; Evangelatos, Gregory P.; Haritos, A.A.; Voelter, Wolfgang

doi:10.1016/0022-1759(92)90152-j

Cited by 22 publications

(12 citation statements)

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“…We verified the specificity of the ELISA in experiments that showed that high concentrations of another h thymosin protein, Th16/ ThNB [8], which is 90% identical to Th15, were not detected by this assay. Th15 levels recovered from a control urine sample spiked with increasing amounts of synthetic Th15 protein averaged 94%, a level equivalent to those observed in other h thymosin assays [16,17,22,23]. Furthermore, this new ELISA achieved the reliability (demonstrated by the reproducibility of the standard curves) and the speed necessary for testing large numbers of clinical samples.…”

Section: Discussionmentioning

confidence: 60%

“…The positions of antibody control, GST-Th15 fusion protein and Th15 are indicated with arrows. Table 4 Demographic and clinical parameters for controls and patients with prostate cancer contrast, antisera raised against intact Th4 or Th9 protein cross-react strongly with other h thymosins, whereas the amino-terminal fragments yield high-titer antiserum with minimal cross-reactivity [16,17,21]. In our study, the aminoterminal fragment (1-12) of Th15 yielded a weak response in the hens and rabbits.…”

Section: Discussionmentioning

confidence: 72%

“…This ELISA relies on the competition for antibody binding between Th15 protein in solution and the Th15 protein immobilized on the microwell surface. The synthetic Th15 protein produced a standard dose-response curve with a useful range of 2.5-625 ng/mL (0.05-30.4 pmol/assay), a sensitivity and range better than those of previous h thymosin ELISAs described in the literature [11,[16][17][18]. We verified the specificity of the ELISA in experiments that showed that high concentrations of another h thymosin protein, Th16/ ThNB [8], which is 90% identical to Th15, were not detected by this assay.…”

Section: Discussionmentioning

confidence: 97%

“…5) and these concentrations were used in the subsequent experiments. Based on the findings of previous studies [16][17][18], we used a 2-h incubation period at 378C, which enabled most antibodies to find their target without compromising the speed of the assay. These conditions produced satisfactory optical density values with minimal background noise and good reproducibility.…”

Section: Standard Curve: Elisa Sensitivity and Specificitymentioning

confidence: 99%

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Development of a sensitive and specific enzyme-linked immunosorbent assay for thymosin β15, a urinary biomarker of human prostate cancer

Hutchinson

Chang

Becker

et al. 2005

Clinical Biochemistry

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Section: Discussionmentioning

confidence: 60%

Section: Discussionmentioning

confidence: 72%

Section: Discussionmentioning

confidence: 97%

Section: Standard Curve: Elisa Sensitivity and Specificitymentioning

confidence: 99%

See 2 more Smart Citations

Development of a sensitive and specific enzyme-linked immunosorbent assay for thymosin β15, a urinary biomarker of human prostate cancer

Hutchinson

Chang

Becker

et al. 2005

Clinical Biochemistry

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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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Comparative evaluation of four trityl‐type amidomethyl polystyrene resins in Fmoc solid phase peptide synthesis

Zikos

Livaniou

Leondiadis

et al. 2003

Journal of Peptide Science

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Four trityl-type (i.e. non-substituted trityl-, o-Cl-trityl-, o-F-trityl- and p-CN-trityl-) amidomethyl polystyrene resins were evaluated comparatively, in terms of the stability of the trityl-ester bond in slightly acidic dichloromethane solutions, and the p-CN-trityl-amidomethyl polystyrene resin was found to be the most stable of them. The above resins were applied, in parallel with Wang benzyl-type resin, well known for its stability in mild acidic conditions, to the Fmoc solid phase synthesis of the 43-amino acid residue long bioactive peptide thymosin beta-4. Independent of their differences in acid sensitivity, the resins seemed to function equally well under the conditions used, since pure thymosin beta-4 was obtained with a final yield of approximately 30% from each resin. The trityl-type amidomethyl polystyrene resins were also applied, in parallel with the Wang resin, to the Fmoc solid phase synthesis of a bioactive peptide containing proline at its C-terminus, i.e. the N-terminal tetrapeptide of thymosin beta-4, AcSDKP. In this case, the best yield (87%) was obtained with the o-Cl-trityl-amidomethyl polystyrene resin, which may be the resin of choice, of those studied, for the Fmoc solid phase peptide synthesis.

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A thymosin β4 ELISA using an antibody against the N terminal fragment thymosin β4[1–14]

Cited by 22 publications

References 12 publications

Development of a sensitive and specific enzyme-linked immunosorbent assay for thymosin β15, a urinary biomarker of human prostate cancer

Development of a sensitive and specific enzyme-linked immunosorbent assay for thymosin β15, a urinary biomarker of human prostate cancer

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

Comparative evaluation of four trityl‐type amidomethyl polystyrene resins in Fmoc solid phase peptide synthesis

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