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Evaluation of antibody quality for Endoglin detection in liver
Kejzlarová, Eliška ; Svobodová, Zuzana (advisor) ; Tripská, Katarína (referee)
The diploma thesis deals with endoglin, a transmembrane glycoprotein that can be cleaved to form so-called soluble endoglin, which then circulates in the blood. The theoretical part of the thesis is devoted to cardiovascular and liver diseases related to changes in the expression or level of endoglin in the blood and summarizes current knowledge about endoglin and its relation to various diseases. The experimental part of the thesis describes the selection of a suitable antibody against endoglin intended for western blot (WB). We have four anti-endoglin primary antibodies available from three manufacturers. The purpose was to test the antibodies and determine, which one had the best ability to detect endoglin in mouse tissue samples such as liver or aorta on WB. Affiblot was used to verify their ability to bind endoglin and also to compare their affinities to this protein. Newly developed patented dot-blot-based device. The binding of the used antibodies to endoglin was verified using the WB method and then the results of both methods were compared. The obtained results showed that the Abcam antibody has the most suitable parameters regarding specificity and sensitivity for the identification of endoglin in mouse tissue on WB. Keywords: endoglin, soluble endoglin, NASH, cirrhosis, non-alcoholic...
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Antibody derivatives for the detection of human glutamatecarboxypeptidase II
Bělousová, Nikola ; Bařinka, Cyril (advisor) ; Pavlíček, Jiří (referee)
Prostate cancer is one of the most common human malignancies and, consequently it is critical to develop appropriate diagnostic and therapeutic tools. Glutamate carboxypeptidase II (GCPII) is currently being considered one of the most important prostate cancer markers due to its tissue- specific expression. Whereas in healthy prostatic tissue the expression levels of GCPII are low, the transformation into the tumor is associated with the substantial increase of GCPII expression, with the highest levels observed in androgen-independent metastatic tumors. GCPII is thus considered a promising marker for early phase as well as advanced metastatic stages of prostate cancer. Current research is focused on the development of highly sensitive and specific reagents that allow detection of small amounts of GCPII, for example in early stages of cancer. Antibody derivatives are promising molecules for this purpose because they have high affinity and specificity and minimum negative side effects. Protein engineering is a prefered approach for preparation of various antibody molecules that differ in size, binding properties, stability, solubility, and production means. Different types of derivatives are being developed for medical needs such as in vitro diagnosis, therapy, and in vivo imagingSmall molecular...
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Formation of anti-hCMV immunity
Písková, Lucie ; Žůrková, Kamila (advisor) ; Fraiberk, Martin (referee)
The human cytomegalovirus (HCMV) is an ancient double-stranded DNA virus that belongs to subfamily β Herpesvirinae and is ubiquitous in human population. Like other herpesviruses infections, the HCMV primary infection leads to a lifelong latency and the virus reactivates from latency periodically through the whole life. The transmission of this virus depends on direct contact among people. The primary HCMV infection is usually asymptomatic in immunocompetent individuals but the reactivated virus is a major cause of diseases in immunocompromised individuals including AIDS patients and allo-graft transplant recipients. This work represents a report about HCMV and its coexistence with the host immune system. The thesis also contains information about complications caused by HCMV in patients who underwent solid-organ or hematopoietic stem cell transplantation.
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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 possibility of determination of HIV antibodies avidity for early infection detection
Diničová, Michaela ; Janďourek, Ondřej (advisor) ; Jílek, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Kralove Department of Biological and Medical Sciences Study program: Specialist on Laboratory Methods Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title of diploma thesis: The possibility of determination of HIV antibodies avidity for early infection detection The determination of early HIV infection in the Czech Republic is based on a combination of results of laboratory examination, clinical examination, and information from an epidemiological report. The identification of patient in the acute phase of the disease allows to search for targeted contact to reduce further spread of HIV, as patients in the early phase of the disease are the riskiest for transmission of infection. One of the laboratory markers of the acute phase of infection is low avidity of antibodies, which expresses the strength of bonds between antigen and antibody. The determination of avidity is based on fact that avidity in recent infections is low because antibodies do not bind tightly enough to antigen and avidity increases when the immune response matures. Quantitatively, it is expressed by the avidity index, which determines what part of the antibodies (%) will remain bound in complex with the antigen after incubation with chaotropic agent, in our case with guanidine...
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Enrichment techniques employed in phosphoproteomics
Fíla, Jan ; Honys, David (advisor) ; Zdráhal, Zbyněk (referee)
Reversible protein phosphorylation represents one of the most rapid and dynamic posttranslational modifications. Phosphorylation plays a key role in many cellular processes, e.g. cell division, signal transduction, cytoskeleton dynamics, translation regulation, protein targeting and metabolism. Unfortunately, the importance of phosphoproteins is often not mirrored in their abundance so enriching strategies are mostly inevitable. The enrichment can be performed either at the stage of phosphoproteins or at the stage of phosphopeptides. Alternatively, a combination of both strategies can be used. Most enriching strategies are based on chemical modifications, affinity of phosphates to positively-charged chromatography matrix or antibodies. Every method used has its advantages as well as disadvantages. Moreover, satisfactory coverage of phosphoproteome is very often reached by combination of several methods. In the past five years many protocols have been improved or alternatively new approaches have appeared. So in this bachelor thesis, the available enrichment strategies are reviewed. Key words: phosphoproteomics, phosphoprotein enrichment, phosphopeptide enrichment, IMAC, MOAC, titanium dioxide TiO2, aluminium hydroxide Al(OH)3, antibodies
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Determination of pepsinogens using IgY and IgG
Kulhavá, Lucie ; Pacáková, Věra (advisor) ; Tichá, Marie (referee)
A decreased concentration of pepsinogen A in serum was found to be a marker of gastric cancer, similarly as a low ratio of pepsinogen A to pepsinogen C. In the present study we have compared properties of immunoglobulin fraction isolated from the egg yolks after immunization of laying hens with pepsinogen isolated from porcine gastric mucosa with those of present in rabbit antiserum obtained after the animal immunization with the same antigen. The characteristics of chicken antibodies against porcine pepsinogen and the comparison with rabbit antibodies raised against the same antigen was carried out using the following methods: ELISA, affinity chromatography on immobilzed antigen and bovine serum albumin, SDS and native electrophoresis and MALDI-TOF MS/MS. While the rabbit specific antibodies interacted with the used antigen and only slightly with bovine serum albumin and there was a diference between pre-immune IgG and specific IgG, in the case of chicken antibodies IgY it did not work. No diference was observed between ELISA tests performed with pre-immune serum and the serum after immunization with porcine pepsinogen and a high interaction of IgY with bovine serum albumin in pre-immune serum and specific IgY after the immunization were detected. Similar results were obtained in experiments with...
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SARS-CoV-2 in Care Home Břevnice
Myslivcová, Lenka ; Andrýs, Ctirad (advisor) ; Jankovičová, Karolína (referee)
The aim of this diploma thesis was to describe information about SARS-CoV-2 virus and coronavirus disease (COVID-19), to evaluate the course of the disease in the Home with a special regime in Břevnice, in which the epidemic took place at the beginning of the first wave of COVID-19 pandemic in spring 2020. Another goal was to perform an antibody analysis and evaluate the obtained data. I divided the diploma thesis into three main parts: theoretical, experimental and discussion. In the theoretical part, I worked with the literature and described information related not only to COVID-19, but also to other serious infections caused by human coronaviruses. In the experimental part, which I performed in the immunological laboratory of the Department of Joint Laboratories at Havlíčkův Brod Hospital, I dealt with the issue of the clinical course of the disease, the severity of the disease and possible consequences after the infection. I also dealt with methods for the determination of antibodies, the principles of which are described in Chapter 4.5. The diagnostic methods and procedures used are described in Chapter 5.3 Laboratory Assays. From the obtained data, I prepared graphs and tables and processed data on the clinical course of the infection in the Břevnice Home, both for the clients and the...
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Immune reactions induced by SARS-CoV-2 infection
Krausová, Kateřina ; Šmahel, Michal (advisor) ; Šroller, Vojtěch (referee)
Coronavirus disease 2019 (COVID-19) pandemic caused by newly discovered Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes severe health and economic problems all over the world. The disease severity depends mainly on the host's immune response to SARS-CoV-2. This virus uses many mechanisms for escape from the host's immune system. The major evasion mechanisms include suppression of interferon production at the early phase of infection, exhaustion of natural killer cells and induction of a cytokine storm. After the innate immune response, mechanisms of adaptive immunity join the defense against the virus. Patients with severe cases have a significant reduction in the amount of both helper CD4+ T cells and cytotoxic CD8+ T cells. On the contrary, these patients have an increased level of antibodies. Even though there have been many findings about immune reactions to SARS-CoV-2 in the year after its discovery, there are still many unknowns. Vaccines, which are successful at preventing COVID-19, have been developed in a short time. However, an important remaining question for further research is the longevity of immune memory after vaccination or after suffering from COVID-19.
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Transmission and epidemiology of visceral leishmaniasis
Spitzová, Tatiana
Visceral leishmaniasis (VL) is widespread disease caused by protozoa Leishmania donovani and Leishmania infantum. Human visceral leishmaniasis caused by Le. donovani in India is considered an anthroponosis, however in East Africa, the role of animals as reservoirs remains unclear. The first part of this thesis demonstrated natural Leishmania infection in wild rodents and bats in Ethiopia. Overall, 8.2% rodents and 4.9% bats were positive for Leishmania spp. Subsequent sequencing revealed that 10% of Leishmania-positive rodents were infected by parasites from Le. donovani complex, on the other hand, no Le. donovani DNA was detected in bats. All Le. donovani-positive rodents were captured in the localities of southwest Ethiopia where human VL cases have been reported and potential sand fly vectors occur. Our findings indicate that rodents are likely to play a role in VL transmission in Ethiopia. During blood feeding, sand flies inoculate into the host skin immunogenic salivary proteins which elicit species specific antibody response. Anti-saliva antibodies could be used as a marker of host exposure to sand flies and, in leishmaniasis endemic areas, also as risk markers of Leishmania infection. In order to find out if the domestic animals (dog, goat, cow, and donkey) from north and northwest Ethiopia...
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