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Indirect analysis of tumor markers via Tag-LIBS with the use of magnetic microbeads
Bačová, Tereza ; Faruzelová, Anna (referee) ; Zikmundová, Eva (advisor)
This bachelor’s thesis is focused on the indirect detection of the prostate cancer biomarker using the Tag-LIBS method. The determination of the biomarker was carried out using photon-upconversion nanoparticles, which, along with the biomarker, are part of the upconversion-linked immunosorbent assay, with magnetic microparticles as the solid phase. The use of magnetic microparticles as the solid phase offers numerous advantages, such as the separation of analyte using an external magnetic field and their use for internal standardization. The Tag-LIBS method was optimized using a suitably chosen double-pulse arrangement, which exhibited higher signal intensity and a lower limit of detection for the biomarker compared to the single-pulse setup. Furthermore, a comparison was made between the upconversion-linked immunosorbent assay using magnetic microparticles as the solid phase and upconversion-linked immunosorbent assay on a microtiter plate. The optimization of the Tag-LIBS method was also aided by the possibility of performing internal standardization. Clinical samples were also analyzed, and the obtained results were compared with the results from upconversion scanning, which is commonly used for detecting the upconversion-linked immunosorbent assay.
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Application of flow cytometry for multiplex analyses in clinical biochemistry
Babušíková, Lucie ; Tremlová,, Bohuslava (referee) ; Beňovská,, Miroslava (advisor)
This thesis discusses the technique of flow cytometry for multiplex analysis and its use in conjunction with imunochemical methods. As part of this work was carried out clinical studies dealing with secondary prevention of myocardial infarction and atherosclerosis in 186 pacientů. In this time represents myocardial infarction worldwide civilizational problem. A number of possible parameters for monitoring atherosclerosis in the world is still an unresolved issue. In the practical part of this work we performed an analysis using Luminex xMAP technology for new parameters (adiponectin, resistin, osteopontin) to predict atherosclerotic disease associated with myocardial infarcion. Also we wanted to see how these parameters are changed in patients after increasing the dose of therapeutic drugs.
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Implementation and optimization of ELISA method
KOPAČKOVÁ, Tereza
Enzyme-Linked ImmunoSorbent Assay or ELISA is a method that has a wide application, especially in immunology. Antigens and antibodies can be detected by the method. The basic principle of the method is the reaction of antigen and antibody and the subsequent formation of an immunocomplex. The immunocomplex is detected by a conjugate that causes a color change of the substrate in the presence of antigen or antibody in the test sample. The ELISA test is a simple method important fot the diagnosis of various diseases or laboratory research. This method is a special type of enzyme immunoassay. The bachelor thesis is focused on the general implementation and optimization of the ELISA method. The aim of the theoretical part of the work is a general introduction to immunological methods. The division of individual types of ELISA tests, including the principles and use in practice, is elaborated in detail. In the practical part, the ELISA method is introduced and optimized using a DYNAREAD photometer for reading 96well microtiter plates and AlaDYN software. In this way, a total of 8 test samples of sera are examined and subsequently the obtained data are processed. The test measurement was performed using the QuatiVac ELISA test kit and human IgG antibodies against SARS-CoV-2 in serum were determined. The peaks of SARS-CoV-2 spike proteins expresses recombinantly in the human cell line HEK 293 were used as antigens. The measurement was performed using a DYNAREAD instrument and data processing was performer using AlaDYN software. 6 calibrators, a positive control, a negative control and then individual serum samples were measured. The measurement results are 5 serum samples in which IgG class anti-SARS-CoV-2 antibodies were detected and 3 samples in which no antibodies were detected.
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Specific prion protein antibodies characterisation and use in diagnostic
Šafaříková, Eva ; Holada, Karel (advisor) ; Mareš, Michael (referee) ; Živný, Jan (referee)
Transmissive spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrPTSE ) in brain. PrPTSE is at present the only specific biochemical marker of human and animal TSEs. Diagnostic tests are based on the detection of PrPres after proteinase K digestion of brain homogenate using Western blot or on the immunohistochemistry of fixed brain tissue, which are both difficult and time consuming. In this work we focused on development of a new type of tests based on PrP detection without need of proteinase K digestion. As deposits of PrPTSE remain in the body for a long time, there is a substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrPTSE may have a potential to serve as a diagnostic marker. We prepared monoclonal antibodies specific for carboxymethyl lysine/arginine modified prion protein. Bacterially expressed and purified recombinant human prion protein (rhPrP) was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of laboratory mice and hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of 4...
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Photon-upconverting nanoparticles as a novel background-free label in immunoassays
Farka, Z. ; Hlaváček, Antonín ; Mickert, M. J. ; Skládal, P. ; Gorris, H. H.
Photon-upconverting nanoparticles (UCNPs) have become an attractive label in immunoassays because their anti-Stokes luminescence can be excited by the NIR laser and detected in the VIS region without optical background interference. Further advantages of UCNPs include good photostability, large anti-Stokes shifts, and multiple narrow emission bands that can be used for multiplexed detection. We have developed a competitive upconversion-linked immunosorbent assay (ULISA) for detection of the pharmaceutical diclofenac (DCF) in surface waters. Silica-coated UCNPs (50 nm in diameter) with carboxyl groups on the surface were synthesized and conjugated with the secondary anti-IgG antibody. The structure and monodispersity of the nanoconjugates was studied by TEM and agarose gel electrophoresis. Using a highly affine anti-DCF primary antibody, the optimized ULISA provided a detection limit of 50 pg·mL−1.
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Specific prion protein antibodies characterisation and use in diagnostic
Šafaříková, Eva ; Holada, Karel (advisor) ; Mareš, Michael (referee) ; Živný, Jan (referee)
Transmissive spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrPTSE ) in brain. PrPTSE is at present the only specific biochemical marker of human and animal TSEs. Diagnostic tests are based on the detection of PrPres after proteinase K digestion of brain homogenate using Western blot or on the immunohistochemistry of fixed brain tissue, which are both difficult and time consuming. In this work we focused on development of a new type of tests based on PrP detection without need of proteinase K digestion. As deposits of PrPTSE remain in the body for a long time, there is a substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrPTSE may have a potential to serve as a diagnostic marker. We prepared monoclonal antibodies specific for carboxymethyl lysine/arginine modified prion protein. Bacterially expressed and purified recombinant human prion protein (rhPrP) was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of laboratory mice and hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of 4...
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Application of flow cytometry for multiplex analyses in clinical biochemistry
Babušíková, Lucie ; Tremlová,, Bohuslava (referee) ; Beňovská,, Miroslava (advisor)
This thesis discusses the technique of flow cytometry for multiplex analysis and its use in conjunction with imunochemical methods. As part of this work was carried out clinical studies dealing with secondary prevention of myocardial infarction and atherosclerosis in 186 pacientů. In this time represents myocardial infarction worldwide civilizational problem. A number of possible parameters for monitoring atherosclerosis in the world is still an unresolved issue. In the practical part of this work we performed an analysis using Luminex xMAP technology for new parameters (adiponectin, resistin, osteopontin) to predict atherosclerotic disease associated with myocardial infarcion. Also we wanted to see how these parameters are changed in patients after increasing the dose of therapeutic drugs.
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Search for new biomarkers for screening and early diagnosis of cancer
KALČÍKOVÁ, Kateřina
Thesis on the topic "Search of New biomakers suitable for screening and early diagnostics of cancer" is dedicated to the issue of cancer, a cancer of the breast, prostate, ovarian and after the new findings out as melanom determination of tumor markers and their implications for clinical practice. Their aim is skouted up biomaker suitable for screening and early diagnostics of carcinoma( cancer ) of the breast, ovarian, prostate and malignant melanoma with utilization of various kinds of immunoassay. The theoretical part gives a brief and comprehensive overview of the epidemiology of selected tumors and tumor markers used and their qualities. In the methodological part are given principles applied iminoanalytics methods a list of the statistical methods and a detailed description of the groups of patients. Results section contains tables with results of biomarkers diveded by sort of avalueted tumors. In the discussion are then analyzed facts that follow from the results. It discussed the process and actualy status of searching convenients biomekers and their combinations that can distinguish a population of benign tumors of the population with malignant tumors and markers which are able to distinguish healthy population from the population with malignant tumors of the breast, ovary, prostate and melanoma. This work may serve as a source of knowledge and results to continue in search of such marker that could be used for screening and early diagnosis of cancer and so to improve the prevention of these illneses in the population.
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