|
|
Preparation and characterization of antipeptide antibodies for immunodetection of cytochromes P450
Mácová, Iva ; Hodek, Petr (advisor) ; Koblas, Tomáš (referee)
The cytochromes P450 are enzymes participating in metabolism of endogenous and exogenous compounds. Their substrates include also carcinogens which may initiate carcinogenesis after activation by CYP450. Inductors of these enzymes are also chemopreventive compounds which are very popular and recommended in current time. Thus, studying of the effect of the chemopreventive compounds on cytochromes P450 induction and cancer development is of a high clinical importance. The CYPs are most commonly found in the liver. However, there are forms that have not been detected in any human healthy tissue but their overexpression was observed in tumors. For this reason, they could serve for diagnosis and prognosis of cancer. Among these cytochromes are CYP2S1 and 2W1 which can be prognostic markers of colorectal cancer. Therefore, it would be opportune to have some tools for these enzyme detection. One option is immunodetection of cytochromes P450 by Western blot using the specific antibodies. Today mammalian antibodies (IgG) are the most widely used but antibodies isolated from egg yolk (IgY) become popular mainly due to the large number of undisputed advantages. For the preparation of the peptide immunogen, suitable peptide sequences were selected from CYP2S1 and 2W1 primary structure. The synthesized peptides...
|
|
|
Development of antibacterial antibodies for cystic fibrosis patients
Vašková, Michaela ; Hodek, Petr (advisor) ; Nosková, Libuše (referee)
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene (CF transmembrane conductance regulator). These mutations result in absent or defective CFTR chloride channel function. The susceptibility to bacterial respiratory infections due to the accumulation of thickened mucus and altered glycosylation in lungs is typical for this disease. Bacteria Pseudomonas aeruginosa (PA) is a major cause of these infections. Among other virulent factors, the pathogenicity of these bacteria is caused by fucose-specific PA-IIL lectin which plays a role as an adhesin. The effect of anti-PA-IIL egg yolk antibodies and multivalent fucose-based PA-IIL inhibitors on PA adherence to lung epithelial cells was studied in this work. Chicken antibodies were isolated from egg yolks before and after immunization with antigen PA-IIL. Specific anti-PA-IIL antibodies were obtained by affinity chromatography using a column with an immobilized PA-IIL. Reactivity of IgY was verified by ELISA. The presence of PA-IIL in the bacterial culture of Pseudomonas aeruginosa (PAK, ST 1763) and the ability of antibodies to recognize this bacterial lectin were verified by Western blotting followed by immunodetection. Appropriate culture conditions have also been found for the expression of this lectin. The...
|
|
|
Preparation and characterization of antipeptide antibodies for immunodetection of cytochromes P450
Mácová, Iva ; Hodek, Petr (advisor) ; Koblas, Tomáš (referee)
The cytochromes P450 are enzymes participating in metabolism of endogenous and exogenous compounds. Their substrates include also carcinogens which may initiate carcinogenesis after activation by CYP450. Inductors of these enzymes are also chemopreventive compounds which are very popular and recommended in current time. Thus, studying of the effect of the chemopreventive compounds on cytochromes P450 induction and cancer development is of a high clinical importance. The CYPs are most commonly found in the liver. However, there are forms that have not been detected in any human healthy tissue but their overexpression was observed in tumors. For this reason, they could serve for diagnosis and prognosis of cancer. Among these cytochromes are CYP2S1 and 2W1 which can be prognostic markers of colorectal cancer. Therefore, it would be opportune to have some tools for these enzyme detection. One option is immunodetection of cytochromes P450 by Western blot using the specific antibodies. Today mammalian antibodies (IgG) are the most widely used but antibodies isolated from egg yolk (IgY) become popular mainly due to the large number of undisputed advantages. For the preparation of the peptide immunogen, suitable peptide sequences were selected from CYP2S1 and 2W1 primary structure. The synthesized peptides...
|
|
|
Humoral response of specific hosts to bird schistosome antigens
Turjanicová, Libuše ; Mikeš, Libor (advisor) ; Kolářová, Libuše (referee)
This thesis focuses on humoral immune response of specific hosts to antigens of various developmental stages of bird schistosomes T. regenti and T. szidati, and follows up on previous research of antibody response in non-specific hosts (mouse, human). Sera of experimentally infected and hunted-down wild ducks were examined using the ELISA and western blot methods. The sera samples were taken in predefined intervals. Results of the ELISA analysis show the process of humoral immune response after infection by bird schistosomes. The level of specific antibodies IgY against homogenate of T. regenti cercariae increased significantly 20 d.p.i. in ducks infected by T. regenti. Such reaction wasn't observed in ducks infected by T. szidati. Slight changes in level of specific antibodies IgM against T. szidati cercariae homogenate were observed 10 d.p.i. only in fully immunocompetent ducks and in reinfected ducks. Examination of hunted-down wild ducks didn't prove infection by bird schisosomes; this conclusion was confirmed by results of the ELISA analysis. IgY antibodies from ducks infected by T. regenti demonstrated strong reactions with 2 antigens in ranges 49-47 kDa and 47-45 kDa. Other reactions, which were recognized, have not been observed in all specimen. An Western blott with homogenate from 7 days...
|
guest ::
