Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The AT-tailing method is a labelling technique that utilises oligo(dA-dT)-dependent signal amplification. In this study, a new immunohistochemical application of the immunoAT method was developed. This method uses an oligo(dA-dT)-conjugated primary antibody (direct immunoAT method) or an oligo(dA-dT)-conjugated secondary antibody (indirect immunoAT method). Fifteen-base oligo(dA-dT)-conjugated antibodies (IgG-ATs) were prepared in advance by conjugating maleimide-activated oligo(dA-dT) to IgG via free sulfhydryl residues that had been introduced on the surface of IgG using Traut's reagent. Following the reaction with the target antigen and the IgG-AT, oligo(dA-dT) was elongated by DeltaTth DNA polymerase in the presence of dATP, dTTP and biotinylated dUTP, consequently labelling the antigen-antibody complex with a large amount of biotin. To initially evaluate the immunoAT method, the presence or absence of prion protein (PrP(sc)) was determined in formalin-fixed and paraffin-embedded sections of the medulla oblongata of cattle which had been under active surveillance for bovine spongiform encephalopathy. Sections were examined using direct and indirect immunoAT methods and the EnVision+ system (Dako) under conditions that were identical except for the differing IgG-AT and AT-tailing methods. PrP(sc) detection was consistent using all three methods. The clearest signals were obtained using the indirect immunoAT method, suggesting significant potential for this method.
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PMID:ImmunoAT method: An initial assessment for the detection of abnormal isoforms of prion protein in formalin-fixed and paraffin-embedded tissues. 2015 61

Heparinoids are the starting material for sulodexide production, a drug used as intravenous anti-coagulant, as an alternative to heparin. The origin determination in the starting material for sulodexide, heparin, and derivatives is crucial for safety (including the impact related to bovine spongiform encephalopathy) and efficacy of the final products. Therefore, European countries have decided to approve the production of heparin only from porcine intestinal mucosa. PCR (polymerase chain reaction) methods are available to evaluate the origin species of crude heparin, during heparin production process, while they lack for the same analysis in heparinoids during sulodexide manufacturing processes. Notably, two main critical issues occur during the origin determination by using PCR for heparinoid analysis: first, heparin has been known to inhibit DNA polymerase activity and, second, the DNA amounts are very low in these samples. To overcome these critical issues, our proposed method is based on two fundamental steps, the DNA concentration by glycogen treatment and DNA purification, which occur before and after DNA extraction, respectively. Finally, by applying real-time PCR, we amplify three specific DNA sequences of ruminant species (bovine, ovine, and caprine), to assess possible contamination, and one from swine, to confirm the origin species. To date, such a method is the only one that determines origin species by PCR for heparinoids that guarantee quality, safety, and traceability of heparin-derived pharmaceutical products. In conclusion, our proposed method is an alternative to nuclear magnetic resonance and ELISA methods, because real-time PCR offers significant advantages in sensitivity, specificity, and robustness. Graphical Abstract.
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PMID:Highly sensitive real-time PCR method to identify species origin in heparinoids. 3180 77