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Lecturer(s)
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Korecká Lucie, doc. RNDr. Ph.D.
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Slováková Marcela, doc. Mgr. Ph.D.
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Course content
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Teaching takes place in blocks. Seminar topics 1. Enzyme immunoassay - competitive and non-competitive methods, RIA and EIA methods, automatic immunochemical analyzers. 2. Methods of preparation of monoclonal and polyclonal antibodies. 3. Methods of non-specific isolation and purification of immunoglobulins and antigens - gel permeation chromatography, ion exchange chromatography, centrifugation and filtration methods, fractional precipitation, dialysis. Methods of specific isolation and purification of immunoglobulins and antigens - basics of affinity chromatography, immunomagnetic separation. 4. Electromigration methods - electrophoretic separation in immunochemical analysis, staining techniques, blotting techniques, densitometric evaluation, isoelectric focusing, multidimensional methods - 2D ELFO. 5. Types of antigen-binding molecules of the antibody base, new methods of gene and protein engineering, techniques for searching antigen-binding molecules. Tasks of laboratories Heterogeneous competitive enzyme immunoassay (ELISA) - quantification of casein protein. Heterogeneous non-competitive ELISA - optimization of parameters for the development of a diagnostic test. Determination of avidity of polyclonal antibodies by modified ELISA method. Basics of bioaffinity chromatography - isolation and purification of IgG antibodies from a serum sample on a column with immobilized protein A. Electromigration methods. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE). Verification of purity of IgG fractions isolated using affinity chromatography. Electrochemical detection - immunosensor for antigen detection.
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Learning activities and teaching methods
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Monologic (reading, lecture, briefing), Laboratory work
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Learning outcomes
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The aim of this subject is to develop the skills necessary to become an expert in immunochemical arrays for analysis of biological materials. The general goal is to develop the student in a flexible fashion adapted to the requirements of analytical practice. Laboratory training in a wide range of immunochemical techniques, in their validation and precise interpretation, preparation of reagents and complex kits is also considered part of the educational program.
Student will obtain good knowledge of all methods used for analysis of biological material, clinical material, food, methods based on the biospecific reaction between antigen and specific antibody. Student know how to install the test in laboratory, how to optimize the condition and validate the test for commercial application.
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Prerequisites
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Student should know the basics from biology, biochemistry, microbiology and immunology.
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Assessment methods and criteria
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Written examination
Laboratory protocols (10 %), written test (60 %) followed by oral examination. To take the oral examination, it is necessary to achieve the 60% specified in the written part.
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Recommended literature
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Návody k laboratořím dostupné ve studijní agendě (STAG)..
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Bartůňková, Jiřina. Vyšetřovací metody v imunologii. Praha: Grada, 2005. ISBN 80-247-0691-1.
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Johnstone A, Thorpe R. Immunochemistry in Practice.. Oxford, 1996.
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Phillips TM. Analytical Techniques in Immunochemistry.. New York, 1992.
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