W12 Immunological techniques Flashcards
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
Antibodies or “immunoglobulins (Ig)” are
specialised proteins produced by B cells
Membrane-bound (B cell receptor)
Secreted
Antibodies are an important part of our immune response
Neutralisation
Opsonisation for phagocytosis
Raising antibodies
The process of producing an antibody specific for a target protein
These antibodies have a wide range of applications in immunological techniques
How do we 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
Blood typing
Blood typing is an example of an “agglutination reaction” used to determine blood type
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)
A, B & RhD are antigens that
that elicit immune responses in mismatched donor/recipient blood transfusions
How does the blood 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
Flow cytometry
Flow cytometry is a technology used to analyse the proteins on cells that are in suspension
Often involves the use of commercially produced antibodies that are conjugated to fluorochromes
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
Fluorochrome
A fluorescent molecule that absorbs light of a certain wavelength and in turn emits light of a certain wavelength
How a flow cytometer works
Fluorochrome-conjugated antibodies specific for the target protein are added to the cells
Cells are channelled past lasers that excite the fluorochrome (e.g. blue laser excites FITC which then emits green light and PE emits Red light)
The li`ght emitted from the excited fluorochromes is detected & plotted on a graph
Amount of light emitted = amount of antibody bound to protein = amount of protein expressed by the cell
Flow cytometer applications
Diagnostics - CD4 T cell counts in HIV
Diagnosis of haematological malignancies
Research - cell sorting
Identification & analysis of immune cells
B cell
lymphoma patient
(Reduced T cell & Granulocytes
Increased B cell clonality)