Immunological Techniques in Diagnostics and research Flashcards
What are immunological techniques?
Includes both experimental methods to study the immune system and methods to generate and use immunological reagents as experimental tools
The most common immunological techniques relate to the production and use of antibodies to detect specific proteins in biological samples
Give examples of immunological techniques in diagnostics and research.
Western
blotting
Antibodies
Agglutination
reactions
Flow
cytometry
Confocal
microscopy
IHC
Immuno-Histochemistry
ELISA
What are antibodies?
Antibodies or “immunoglobulins (Ig)” are specialised proteins produced by B cells
- Membrane-bound (B cell receptor)
- Secreted
Describe the antibodies structure.
Antibodies have a Y-shaped structure
-2 × heavy chains & 2× light chains
-2 parts: Fc & Fab fragments
-Fab region is specific for a protein (“antigen”)
-5 different types of heavy chain in mammals generates 5 “isotypes” IgA, IgG, IgD, IgE &
IgM
What are antibodies important for?
Neutralisation
Opsonization for phagocytosis
What is raising antibodies?
The process of producing an antibody specific for a target protein. Thses antibodies have a wide ra
How do you raise antibodies?
- Mice are immunised with the target protein
- B cells are harvested & fused with tumour cells to form hybridoma
- A hybridoma that produces antibody against the target protein is selected & cloned
- The antibodies secreted by the cloned hybridoma are harvested & used in immunological techniques
What is Blood typing?
Blood typing is an example of an “agglutination reaction” used to determine blood type
Describe the ABO system of blood typing.
ABO system of blood typing
- A & B glycoproteins on RBCs
- Four types of blood A, B, AB & O
- Rhesus factor (D protein or RhD) is also a protein on the surface of RBCs (positive or negative)
How does the blood typing test work?
- A blood sample is mixed with antibodies raised against A, B or RhD antigens
- The sample is visually checked for agglutination (blood cells sticking together)
- Agglutination indicates the presence of antigens in the blood sample
What antigens elicit immune response?
A, B & RhD are antigens that elicit immune responses in mismatched donor/recipient blood transfusions
What is flow cytometry?
Flow cytometry is a technology used to analyse the proteins on cells that are in suspension.
What can flow cytometry determine about a cell?
-The cell size and density
-Whether or not a cell expresses a target protein
-The amount of expression of a target protein
-The cells identity
Often involves the use of commercially produced antibodies that are conjugated to fluorochromes.
What is a fluorochrome and how is it involved in flow cytometry?
A fluorescent molecule that absorbs light of a certain wavelength and in turn emits light of a certain wavelength.
Fluorochrome-conjugated antibodies specific for the target protein are added to the cells
