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)
Confocal microscopy differences w/flow cytometer
The cells to be analysed are not in suspension
Used to analyse tissue sections or cells attached to a microscope slide
The light emitted by the fluorochrome-conjugated antibodies is observed under a microscope instead of plotted graphically
Confocal microscopy has the advantag`e of visualising where the protein is on the cell
Confocal microscopy - applications
Mainly research
Identification & analyse cells within tissues
Co-localisation of different antigens
IHC
IHC stands for ImmunoHistoChemistry
Used to show the distribution & localization of antigens in tissue sections using antibody-antigen interactions
How does IHC work?
Thin sections of tissue are cut
Primary antibodies that recognise the target protein are added to the tissue
The antibody-antigen interaction is visualised using chromogenicdetection
A secondary antibody specific for the primary antibody conjugated to horseradish peroxidase (HRP) is added
HRP catalyses the conversion of the chromogen 3,3-diaminobenzidine (DAB) substrate to produce a brown precipitate at the location of the protein
The brown precipitate is then visualised using a light microscope
Example of IHC
IHC is used to stain B-Raf protein in tissue sections from cancer patients and direct eligible for treatment with B-Raf inhibitors
Cancer can be caused by mutations in tumour suppressor or tumour promoter genes
These genes encode proteins that are involved in suppressing or promoting cell division
Mutations in these genes can cause uncontrollable cell division
E.g. BRAF is a gene that encodes B-Raf protein that promotes cell division
Many cancers have BRAF mutations that result in over production of B-Raf and thus uncontrolled cell division
ELISA
Enzyme-Linked ImmunoSorbant Assay
Quantifies the amount of a protein or antibody in liquid samples such as sera or tissue culture supernatants
Four different types of ELISA
Direct
Indirect
Sandwich
Competitive
ELISA applications
Antibody titres in patient serum e.g viral infections such as HIV & Hepatitis B
Detection of bacterial toxins in food such as Escherichia coli O157:H7
Home pregnancy testing detection of human chorionic gonadotropin hormone (HCG) in urine
Research quantification of cytokines/chemokines/growth factors in tissue culture supernatants
Sandwich ELISA
Add capture antibody
Add sample (serum or TC spnt)
Add detection antibody (HRP - horse radish antibody)
Add substrate (TMB - a chromogenic substrate)
Determine optical density
Density of blue solution = amount of protein in sample
Differences in the types of ELISA
Direct ELISA - subsrate + primary antibody conjugate
Indirect ELISA - substrate + secondary antibody conjugate
Sandwich ELISA - substrate + capture antibody
Competitive ELISA - substrate + inhibitor antigen
Western blotting
a technique used to detect proteins
A Southern Blot (Edwin Southern) detects DNA
A Northern blot detects RNA
Western blot involves four steps
Sample preparation
Electrophoresis
Transfer to membrane
Stain for protein of interest
Sample preparation - western blot
Cells are lysed & proteins denatured
Electrophoresis - western blot
Lysates are loaded onto a gel & proteins separated based on size
Transfer to membrane - western blot
Fractionated proteins are transferred onto a membrane
Stain for protein of interest - western blot
The membrane is incubated with a primary antibody specific for the target protein
The membrane is then incubated with a HRP conjugated secondary antibody specific for the primary antibody
A chemiluminescent HRP substrate is added to the membrane
The membrane is exposed to x ray film that “bleaches” when exposed to light
Diagnostic use - western blot
The pathogen is lysed & its proteins are separated on a gel.
The proteins are transferred to a membrane.
The membrane is mixed with patient serum to capture antibodies.
Excess is washed off then secondary antibodies are added to visualise.
eg. Viral infections (HIV)
parasites (spirochete Borrelia burgdorferi that
causes Lyme disease)
Research - western blot
Cell signalling proteins
Mechanism of action for cancer drugs
