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Imunochemická detekce proteinu TNF-α pomocí magnetických částic
Koudelková, Žaneta
This thesis is focused on development and optimization of a method for detection of the cytokine TNF-α by the sandwich ELISA using magnetic particles. These particles were functionalized with carboxylic functional groups to enable the antibody binding. Optimal pH of the MES buffer, EDC concentration, and method of activation were determined by optimizing the conditions of particle activation. In the second part of the thesis, the conditions of the magnetic particle-based sandwich ELISA were optimized. It was discovered that mixing the samples during incubation resulted in a higher number of molecules being captured by the antibodies. The optimal incubation times for the antigen and detection antibody were determined to be 3 hours and 1.5 hours, respectively. The optimized conditions for particle activation and ELISA were used to create a calibration curve of TNF-α standards. Optimized magnetic particle-based ELISA protocol can simplify and enhance the analysis of complex samples like blood serum, urine, or saliva.
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Chicken antibodies as a tool of cytochrome P450 immunodetection
Mácová, Iva ; Hodek, Petr (advisor) ; Koblas, Tomáš (referee)
Cytochrome P450 is an important enzyme which catalyzes a large amount of reactions of endogenous and exogenous metabolism. There are compounds which increase expression of this enzyme and it leads up to the carcinogen activation and cancer formation. It includes much propagated chemopreventive compounds which are consumed abundantly in dietary supplements. It is therefore important to have the methods for determination of cytochrome P450 induction. One of these methods is the cytochrome P450 immunodetection by Western blotting. The detection of proteins on the membrane is done by the specific antibodies mostly gained from mammals. In this bachelor thesis there is the evidence that the antibodies from hen egg serve as well as the mammalian antibodies. The cytochromes P450 1A1 and 1A2 was detected by these egg antibodies after rat exposure to the model chemopreventive compounds β-naphthoflavone.
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Study of selected flavonoids as modulators of cytochrome P450 3A4
Skotnicová, Aneta ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
Flavonoids are secondary metabolites of plants exhibiting a wide range of biological activities. Special attention is paid to them mainly because of their antioxidant properties. Consumption of these natural substances is currently increasing especially in the form of dietary supplements. In addition, these compounds exhibit antiviral, anti-inflammatory or hepatoprotective effects and some of them also exhibit mutagenic and/or pro-oxidative properties. The study of flavonoid interactions with biotransformation enzymes such as cytochromes P450 are important because of their possible interference with the metabolism of drugs or carcinogenesis processes. In this bachelor thesis the influence of selected flavonoid compounds, dihydromyricetin and myricetin, on the expression and activity of cytochrome P450 3A4 was studied. The flavonoid premedication of rats did not resulted in induction of CYP3A4. However, dihydromyricetin and myricetin inhibit CYP3A4. IC50 were determined as 25 M for dihydromyricetin and 24 M for myricetin. Key words: Immunodetection, HPLC, inhibition, induction (In Czech)
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Development, characterization and use of anti-orphan cytochrome P450 antibodies
Hrdinová, Johana ; Hodek, Petr (advisor) ; Koblas, Tomáš (referee)
The cytochromes P450 (P450s) are important enzymes involved in metabolic pathways, which use exogenous and endogenous substances as their substrate for various enzymatic reactions. These enzymes can also use precarcinogens as their substrate and activate them into carcinogens, which leads to a cancer development. If the P450s are induced, the cancer risk increases. Some chemopreventive compounds may induce the P450s and thus be harmful to the human body. Therefore it is necessary to pay enough attention to a study of the mechanism of action of P450s and the influence of the chemopreventive compounds on the activity of cytochromes P450. mRNA expression of most of the P450s isoforms is detected in a number of healthy (nontransformed) tissues, viz. liver, brain, heart, colon, kidney or placenta. Nevertheless there are a few P450s isoforms which mRNAs are expressed at relatively low levels in the nontransformed tissues, whereas the expression in the transformed tissues is significantly higher. One of these P450s is CYP2W1, which can be used as a prognostic marker for colorectal cancer - therefore it is useful to be able to detect a presence of this enzyme in various tissues. A detection of P450s can be accomplished by using a method Western blot. In this method, the immunodetection is achieved by using...
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Chicken antibodies as a tool of cytochrome P450 immunodetection
Mácová, Iva ; Hodek, Petr (advisor) ; Koblas, Tomáš (referee)
Cytochrome P450 is an important enzyme which catalyzes a large amount of reactions of endogenous and exogenous metabolism. There are compounds which increase expression of this enzyme and it leads up to the carcinogen activation and cancer formation. It includes much propagated chemopreventive compounds which are consumed abundantly in dietary supplements. It is therefore important to have the methods for determination of cytochrome P450 induction. One of these methods is the cytochrome P450 immunodetection by Western blotting. The detection of proteins on the membrane is done by the specific antibodies mostly gained from mammals. In this bachelor thesis there is the evidence that the antibodies from hen egg serve as well as the mammalian antibodies. The cytochromes P450 1A1 and 1A2 was detected by these egg antibodies after rat exposure to the model chemopreventive compounds β-naphthoflavone.
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