Konference: 2015 XI. Dny diagnostické, prediktivní a experimentální onkologie
Kategorie: Nádorová biologie/imunologie/genetika a buněčná terapie
Číslo abstraktu: p04
Autoři: Ing. Simona Dostálová; Ing. Kristýna Šmerková; Marcela Vlčnovská; Ing. Vedran Milosavljevic; doc.RNDr. Pavel Kopel, Ph.D.; Mgr. Tomáš Vaculovič, Ph.D.; Ing. Soňa Křížková, Ph.D.; Mgr. Markéta Vaculovičová (Ryvolová), Ph.D.; doc.RNDr. Vojtěch Adam, Ph.D.; doc.Ing. René Kizek, Ph.D.
Magnetic separation employs spherical nanometric or micrometric structures with paramagnetic or superparamagnetic properties for isolation of various interesting compounds , including the cisplatinated (cisPt) DNA. This method can be employed as an easy way of detecting guanine-rich regions in DNA  or to monitor the pharmacokinetics of cisplatin-DNA interactions .
DNA fragment of phage λ xis gene was mixed with cisPt of various concentrations and incubated to form adducts. Dynabeads Protein G particles were used for immunoseparation. The antidsDNA antibodies were bound on the MPs‘ surface according to the manufacturer‘s instructions. CisPt-DNA sample was added and incubated to form complex. AnticisPt- modified DNA antibody was added to the complex and CdTe QDs modified with HWR peptide were bound to this antibody. The fluorescence of the QDs was measured after purification with the excitation wavelength of 360 nm.
Results and conclusions
The reactivity of both used antibodies was tested using dot blot. Anti-dsDNA antibody was able to detect 0.2 nM concentration of DNA with a linear dependence on concentration. This reactivity was not lost after cisplatination of DNA. The reactivity of anti-cisPt-DNA was linearly increased with increasing incorporated platinum in DNA. The capacity of the magnetic particles was 10 μg of antibodies per mg of the particles. The sandwich immunoassay caused 6.6-fold increased fluorescence of QDs labelled anti-cisPt-DNA antibodies compared with isolation of DNA without cisPt. Using this approach, 86% of applied DNA can be detected on the particles.
The authors gratefully acknowledge financial support from the Grant Agency of the Czech Republic (NanoBioTECell P102/11/1068).
- Zitka, O. et al. Preconcentration based on paramagnetic microparticles for the separation of sarcosine using hydrophilic interaction liquid chromatography coupled with coulometric detection. J. Sep. Sci. 37, 465-475, (2014).
- Siddik, Z. H. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 22, 7265-7279, (2003).
- Chen, H. Y. et al. Interaction of electrons with cisplatin and the subsequent effect on DNA damage: a density functional theory study. Phys. Chem. Chem. Phys. 16, 19290-19297 (2014).
Datum přednesení příspěvku: 7. 2. 2017