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Preparation of Monoclonal Antibodies Against VP2 Protein of Human Polyomaviruses
Vochyánová, Klára ; Drda Morávková, Alena (advisor) ; Růžičková, Šárka (referee)
Aim of this diploma thesis was to prepare two protein antigens and two monoclonal antibodies, all based on VP2 minor protein of human polyomaviruses BK virus and Merkel Cell Polyomavirus. One monoclonal antibody was being prepared against unique part of VP2 protein (N-terminal epitope, not present in VP3 protein). A cell line producing such monoclonal antibody has never been established before due to low immunogenicity of the epitope. Our approach was successful in terms of mouse immunization, however, serious problems with hybridoma line stability appeared later during the preparation process. Preparation of antibody targeted to the sequence of VP2 protein of Merkel Cell Polyomavirus was another aim of this thesis. Mouse immunization and hybridoma fusion were performed successfully. After four rounds of cloning in order to purify an established clone, nine clones were cultivated in larger scale. This cultivation probably led to diminished antibody specificity and loss of production ability in most of the hybridoma cells. One more cloning should give rise to an established clone with sufficient production. Two preparations of protein antigens were performed in two expression systems. DNA encoding C-terminally truncated protein VP2 of BK virus fused with His-tag was cloned into a vector suitable for...
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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...
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Development of tool for passive immunization of cystic fibrosis patients
Pacholíková, Lenka ; Hodek, Petr (advisor) ; Švédová, Martina (referee)
Cystic Fibrosis (CF) is one of the most common heriditary diseases. This congenital condition, caused by CFTR gene mutation, affects gastrointestinal and respiratory system especially. The affection of respiratory system is considered the most serious life-threatening symptom. Pacients suffer from reccurent infections proceeding to the development of chronic inflammation and progressive pulmonary tissue destruction. A typical specific microoganism colonizating pulmonary tissue of thouse suffering from CF is recognised as Pseudomonas aeruginosa. Pulmonary infections caused by this microorganism are the most often cause of death in patients suffering from CF. Antiobiotics are the first-line therapy of this condition currently. Nevertheless, the need to find alternatives occurs due to antibiotics resistance development. Passive immunization by specific hen-egg-yolk antibodies against P. aeruginosa is a possible alternative. An observation of IgY influence on a bacterial adhesion to pulmonary epithelial cells required an appropriate model. In this context an appropriate adhesion testing method based on P. aeruginosa and pulmonary epithelial cells visualisation was searched. At first bacterial cells labelling was tested by CellTracker, resazurin, FITC and consequently PKH 26. P. aeruginosa fluorescent...
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Immunity in hosts repeatedly exposed to sand fies and the effect on pathogen transmission
Pohanková, Lucia ; Kolářová, Iva (advisor) ; Fialová, Anna (referee)
6 Abstrakt During the feeding of infected sand flies are inoculate into the host also sand fly saliva, which can strongly modulate the response of the immune system. If the host is naive, the course of infection is usually worse. In cutaneous leishmaniasis, the lesions developed early, are more destructive and persist longer. The hosts living in endemic areas of leishamniasis and their vector hosts are often exposed to feeding uninfected sand flies. For host are the saliva antigenic and induces specific cellular and antibody responses. This responses induce the protection against leishmania infection and differ for different hosts, attempts were made most frequently in murine and canine models. In humans, as hosts is difficult to monitor developments leishmania infection after previous exposure, because in humans mainly monitors the levels of antibodies, by which we can determine the degree of sand fly bites and the risk of transmission of leishmaniasis. Keywords: Lutzomyia, Phlebotomus, DTH, antibody, IgG,
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The testing of celiac disease in connection with osteoporosis
SLADKÁ, Romana
My Bachelor´s thesis deals with the examination of celiac disease in connection to osteoporosis. The aim of the thesis was to specify the level of antidotes against tissue transglutaminase in the class IgA and IgG in the serum with osteoporosis diagnosed patients, to interpret, based on the results of the study, possible link between osteoporosis and celiac disease as well as to compare the outcome with scientific data. I focused on the present knowledge of osteoporosis and celiac disease in the first part. As for celiac disease, I described its history and characteristics. I also paid attention to diagnostics of celiac disease with the assistance of laboratory serological tests ELISA. I mentioned the free-gluten diet treatment after that. As regards osteoporosis, I described its history, symptoms, forms, perils and complications which the disease causes. I conducted the methodical part of my work in the biochemical haematological laboratory Stafila where I work, with its seat in České Budějovice. As for measurements, I took them in the department of microbiology, where immunologic tests are also taken. I carried out the screening of celiac disease from serum, namely the determination of antidotes against tissue transglutaminase in the class IgA and IgG, with the aid of ELISA - a sandwich method. I took measurements with 58 osteoporosis diagnosed patients. I obtained the sera thanks to the supervisor of my thesis Mrs. Marie Ládová, who is at the head of an osteological outpatient department in České Budějovice. I stated the serological markers of celiac disease with the assistance of enzymatic immunoanalysis conducted on automatic analyzer Nexgen Four, which was provided by the firm Test-Line. I describe the analyzer, its specification, checking and calibration of the gadget together with determination checks in this part of the thesis. Additionally, there is described the principle of the method ELISA- sandwich, which is non-competitive enzymatic immunoanalysis. At the end of the section there is depicted the methodical procedure of my work, the preparation of working solutions and checks, the preparation and dilution of samples for analysis and working procedure for semiquantitative interpretation with positivity index and for quantitative interpretation U/ml. I continued with my own measurements. while respecting standard operations procedures of the laboratory Stafila, a limited liability company. The results of my measurements are processed in the third part of the thesis. The acquired results are given in a table: quantitative interpretation of the antidote against tTg IgA and IgG (U/ml) level in the sera of the osteoporosis diagnosed patients. Then I presented a graph interpreting the proportional relation of the antidote against tTg IgA and IgG levels in the sera of the osteoporosis diagnosed patients. There are included calibration graphs for tissue transglutaminase IgA and IgG and tables of the obtained data of checks for the methods in the results. 3 out of the 58 examined samples of osteoporotic patients were positive, they had the level of antibodies higher than 22 U/ml. One of the samples had marginal figures, which means it had the antidotes against tTg IgA an IgG between 18 and 22 U/ml. Positive results were found with patients born in 1976 and 1978. A patient born in 1962 appeared to have a marginal result. The acquired antidote figures against tissue transglutaminase in the class IgA and IgG are presented proportionally in the graph 1. 3 out of the 58 measured samples were positive, which represents 5%, and 1 sample was marginal, which is 2%. My analysis is based on the availability of the examined material and the potential of our laboratory. Celiac disease is an illness which was not paid much attention to in the past. Hypothesis about the link between osteoporosis and celiac disease has been proved.
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