C8116 Immunochemical techniques

Přírodovědecká fakulta
jaro 2024
Rozsah
2/0/0. 2 kr. (plus ukončení). Ukončení: zk.
Vyučující
prof. Dr. rer. nat. habil. Hans-Heiner Gorris (přednášející)
Garance
prof. Dr. rer. nat. habil. Hans-Heiner Gorris
Ústav biochemie – Chemická sekce – Přírodovědecká fakulta
Dodavatelské pracoviště: Ústav biochemie – Chemická sekce – Přírodovědecká fakulta
Rozvrh
Po 19. 2. až Ne 26. 5. Út 13:00–14:50 B11/335
Předpoklady
C3181 Biochemie I
A completed bachelor's degree is required for attending the course. Additional recommendation: C3181 Biochemistry I Basic knowledge of biology and general chemistry.
Omezení zápisu do předmětu
Předmět je nabízen i studentům mimo mateřské obory.
Mateřské obory/plány
Anotace
The course aims at a fundamental understanding of immunology and an advanced understanding of immunochemical methods and fluorescence microscopy. The main topics are: A) immune system, B) antibody labels, C) immunoassays, D) protein-protein interactions, E) advanced fluorescence microsocpy. The course is complementary to C3002 Nanobiotechnology.
Výstupy z učení
After completing the course, a student will understand - the main players of our immune system and the benefits of getting vaccinated. - what makes antibodies such a terrific biochemical tool. - that immunoassays safe thousands of lifes every day. - how protein-protein interactions hold the key to their function. - what can (and what cannot) be visulized by fluorescence microscopy.
Klíčová témata
    • Introduction: Overview on immunochemical techniques
    • Part A: The immune system and its sharpest weapon: antibodies
    • 1) General introduction to the immune system
    • 2) Innate / adaptive immune system
    • 3) Lymphoid organs
    • 4) B cells, helper/cytotoxic T cells; clonal selection
    • 5) MHC class I and II, T-cell receptor, antigen presenting cells
    • 6) Generation of antibody diversity / affinity maturation
    • 7) Complement system
    • 8) Immunoglobulin superfamily and function of antibody classes IgG, IgM, IgA, IgE
    • 9) Binding site of antibodies: intermolecular forces facilitate antigen binding
    • 10) Antibody affinity vs. avidity
    • 11) Antigen binding: key-lock principle vs. induced fit
    • 12) Antigen determinants: hapten / epitope
    • 13) Raising an immune response in mice
    • 14) Generation of monoclonal antibodies (Milstein/Köhler)
    • 15) Vaccination (active and passive), antisera
    • 16) Vaccination strategies: life/dead vaccine, vectors, mRNA, e.g. Sars-Cov-2
    • 17) Therapeutic antibodies
    • Part B: Antibodies as immunological tools
    • 1) Handling of antibodies
    • 2) Antibody labels (enzymes, fluorophores, NPs, radionuclides, biotin/streptavidin system)
    • 3) Detection systems: Photomultiplier tubes vs. camera systems (spatial resolution)
    • 4) Determination of antibody affinities by surface plasmon resonance (SPR) or quartz crystal microbalance QCM)
    • 5) Limitations of natural antibodies (affinity ceiling, relatively large size)
    • 6) Alternatives: recombinant, humanized, cameloid, single chain antibodies)
    • 7) Phage display for affinity maturation of antibodies in vitro
    • 8) Aptamers (SELEX)
    • 9) Molecularly imprinted polymers (MIPs, “plastic antibodies”)
    • Part C: Immunoassays
    • 1) Definition and key developments of immunoassays
    • 2) Applications of immunoassays (diagnostic, environmental, food safety)
    • 3) Matrix interference (medical, environmental samples) and non-specific binding
    • 4) Definition: sensitivity / limit of detection / limit of quantification
    • 5) Competitive / non-competitive
    • 6) Heterogeneous immunoassays
    • - Direct / indirect / sandwich format
    • - Radioimmunoassay (RIA)
    • - Enzyme-linked immunosorbent assay (ELISA)
    • - Fluorescence immunoassay (FIA)
    • - Electrochemiluminescent / photoelectrochemical immunoassay
    • - Chemiluminescent assay
    • - Time-resolved immunoassays, e.g. “Dissociation enhanced dissociation-enhanced lanthanide fluorescence immunoassay” (DELFIA)
    • - Nanoparticles as detection labels, e.g. “Upconversion-linked immunoassay” (ULISA)
    • - Signal amplification strategies (e.g. Immuno-PCR)
    • 7) Lateral flow assays / test strips
    • 8) Homogeneous immunoassays (“mix and measure”)
    • - Fluorescence polarization immunoassay (FPIA)
    • - Fluorescence resonance energy transfer (FRET)
    • - Proximity ligation assay
    • - Aggregation-based immunoassays
    • 9) Suspension arrays (magnetic beads)
    • 10) Multiplexing
    • 11) Single-molecule / digital immunoassays (Quanterix Inc.)
    • 12) Microarrays (concept of ambient analyte assays)
    • 13) Microfluidic assays
    • 14) From our own work: assay design principles
    • 15) Commercialized immunoassay test kits
    • 16) Immunosensors
    • Part D: Immunoaffinity and other protein-protein affinity techniques
    • 1) Immunoblotting
    • 2) Immune diffusion (Ouchterlony test)
    • 3) Immune precipitation (Nephelometry)
    • 4) Co-immunoprecipitation
    • 5) Affinity chromatography
    • 6) GST pulldown assay
    • 7) Tandem affinity purification: tap tagging
    • 8) Yeast two hybrid system
    • 9) (far) Western blotting
    • Part E: Advanced fluorescence microscopy for (life) cell imaging
    • 1) Repetition of fundamentals in (far-field) optical microscopy
    • 2) Immunocytochemistry and immunohistochemistry
    • 3) Advantages and limitations of immunofluorescent labelling for microscopy
    • 4) Confocal microscopy
    • 5) Single-molecule fluorescence microscopy
    • 6) Multiphoton microscopy
    • 7) Super-resolution microscopy (STED/STORM)
    • 8) Light sheet microscopy
    • 9) Fluorescence-activated cell sorting (FACS)
    • 10) Electron microscopy, Cryo electron microscopy
Přístupy, postupy a metody používané ve výuce
Lecture.
Způsob ověření výstupů z učení a požadavky na ukončení
Oral examination.
Vyučovací jazyk
Angličtina
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