C8116 Immunochemical techniques

Faculty of Science
Spring 2022
Extent and Intensity
2/0/0. 2 credit(s) (plus extra credits for completion). Type of Completion: zk (examination).
Teacher(s)
Dr. rer. nat. habil. Hans-Heiner Gorris (lecturer)
Guaranteed by
Dr. rer. nat. habil. Hans-Heiner Gorris
Department of Biochemistry – Chemistry Section – Faculty of Science
Supplier department: Department of Biochemistry – Chemistry Section – Faculty of Science
Timetable
Tue 10:00–11:50 C05/114
Prerequisites
C3181 Biochemistry I
A completed bachelor's degree is required for attending the course. Additional recommendation: C3181 Biochemistry I Basic knowledge of biology and general chemistry.
Course Enrolment Limitations
The course is also offered to the students of the fields other than those the course is directly associated with.
fields of study / plans the course is directly associated with
Course objectives
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.
Learning outcomes
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.
Syllabus
  • 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

Teaching methods
Lecture.
Assessment methods
Oral examination.
Language of instruction
English
Further Comments
The course can also be completed outside the examination period.
The course is taught annually.
The course is also listed under the following terms Spring 2023, Spring 2024, Spring 2025.
  • Enrolment Statistics (Spring 2022, recent)
  • Permalink: https://is.muni.cz/course/sci/spring2022/C8116