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Lecturer(s)
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Korecká Lucie, doc. RNDr. Ph.D.
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Course content
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Overview of immunochemistry - basic terminology, antigens (selection, structure, function), antibodies (classification, structure, function), reaction antigen-antibody (kinetics, affinity, avidity, specificity, cross-reaction), monoclonal and polyclonal antibodies - preparation, characterization. Isolation of antibodies, purification, characterization, fragmentation, identification. Enzyme immunoassay - homogeneous, heterogeneous, competitive, non-competitive arrangement, qualitative and quantitative data evaluation. Preparation of labelled antigens/antibodies (hapten-protein, hapten-enzyme, antibody-enzyme, radioisotope labelling, fluorochromes). Automatic analysers in immunochemistry. Separation and Analytical methods used in immunochemical laboratories - precipitation, ultrafiltration, spectrophotometry, liquid chromatography, size-exclusion chromatography, ion-pair chromatography, bioaffinity chromatography, electrophoretic methods. Immunoaffinity chromatography - theoretical and practical aspects, antibody-based immunosorbent preparation, antibody selection, immobilization principles, column arrangement, utilization in practise. The use of antibodies in cytology (immunophenotyping) and immunohistochemistry. Immunochemical methods for analysis and identification of peptides, proteins - 2D ELFO, HPLC, CZE, MS, microchip technology. Biosensors in immunochemistry - electrochemical detection of antigen-antibody immunocomplex, labelling, detection methods, miniaturization. Competitive enzyme-based immunoassay, affinity constant evaluation, Scatchard evaluation. Enzyme-linked immunosorbent assay (ELISA): Determination of antibody titre, Quantification of antigen. ELISA: Determination of avidity of polyclonal antibodies with use of chaotropic reagent, optimization of reaction conditions - selection of the best ratio of dilution of antigen and labelled antibody. SDS-PAGE electrophoresis: Separation of proteins, confirmation of molecular weight of immunoglobulins, separation of H and L chains of IgG. Isoelectric focusation. Methods for isolation and purification of immunoglobulins - affinity chromatography, size-exclusion chromatography, ion-pair chromatography, magnetic separation. Comparison of isolation efficiency, product purity. Electrochemical detection of antigen (ovalbumin, ApoE protein) based on antigen-antibody reaction.
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Learning activities and teaching methods
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Monologic (reading, lecture, briefing), Dialogic (discussion, interview, brainstorming), Methods of individual activities, Laboratory work
- Participation in classes
- 39 hours per semester
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Learning outcomes
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Lectures: Overview of immunochemistry - basic terminology, antigens (structure, function), antibodies (structure, function), reaction antigen-antibody (kinetics, affinity, avidity, specificity, cross-reaction), monoclonal and polyclonal antibodies - preparation, characterization, isolation of antibodies. Immunochemical methods - serological methods, immunoanalytical methods with indicators - EIA, RIA, FIA, CLIA, ECLIA. Magnetic beads-based immunoanalytical methods. Optimization and validation of immunoanalytical methods. Electrophoretic methods with immunochemical detection - PAGE, western-blot, dot-blot. Immunosensors for diagnostics. Immunoaffinity chromatography - method arrangement, optimization steps, detection. Laboratories: Immunoagglutination, immunoprecipitation. Enzyme-linked immunosorbent assay (ELISA): Quantification of antigen - non-competitive heterogeneous ELISA, Enzyme-linked immunosorbent assay (ELISA): Quantification of antigen - competitive heterogeneous. Magnetic beads-based ELISA for quantification of antigen. ELISA: Determination of avidity of polyclonal antibodies with use of chaotropic reagent, optimization of reaction conditions of ELISA - selection of the best ratio of dilution of antigen and labelled antibody. Electrochemical immunosensors - electrochemical detection of antigen, labelling, detection methods, miniaturization. Western blot - detection of antigen - 1D SDS-PAGE, semi-dry trans blot turbo, colorimetric and chemiluminescent detection. Densitometric evaluation Aims: to prepare students for work in immunochemical laboratory for analysis of biological samples. To bring brief overview of basic concepts of immunochemical reaction, components. Specific examples using immunochemical methods. Practical implementation of ELISA methods, electromigration methods, western blot and biosensors. Optimization of the ELISA method to gain an overview of how diagnostic tests are developed for routine laboratory diagnostics.
Student obtain good knowledge of all methods used for analysis of biological samples, 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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Students should have basic knowledge of cell biology, biochemistry, immunology and instrumental analytical methods
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Assessment methods and criteria
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Written examination
written test
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Recommended literature
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Ferencik M. Handbook of Immunochemistry.. New York, 1993.
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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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