C8116 Immunochemical techniques

Přírodovědecká fakulta
jaro 2023
Rozsah
2/0/0. 2 kr. (plus ukončení). Ukončení: zk.
Vyučováno prezenčně.
Vyučující
Dr. rer. nat. habil. Hans-Heiner Gorris (přednášející)
Garance
Dr. rer. nat. habil. Hans-Heiner Gorris
Ústav biochemie – Chemická sekce – Přírodovědecká fakulta
Dodavatelské pracoviště: Ústav biochemie – Chemická sekce – Přírodovědecká fakulta
Rozvrh
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
Cíle předmětu
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.
Osnova
  • 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

Výukové metody
Lecture.
Metody hodnocení
Oral examination.
Vyučovací jazyk
Angličtina
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