Practical Knowledge and Application/Immunological Techniques Flashcards
What are immunological techniques?
Immunological techniques include 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.
What are raising antibodies?
Process of producing an antibody specific for a target protein. These antibodies have a wide range of applications in immunological techniques.
ANTIBODIES
- Specialised membrane-bound proteins produced by B cells
- Important for neutralisation and opsonisation for phagocytosis
- 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
How do we raise antibodies?
1) Mice are immunised with the target protein
2) B-cells are harvested and fused with tumour cells to form a hybridoma
3) A hybridoma that produces antibodies against the target protein is selected and cloned
4) The antibodies secreted by the clones hybridoma are harvested and used in immunological techniques
Monoclonal antibody
- Only has one binding site for one specific shape on one specific antigen
What is blood typing?
Example of an ‘agglutination reaction’ used to determine blood type.
A, B and RhD are antigens that elicit immune responses in mismatched donor/recipient blood transfusions.
1) A blood sample is mixed with antibodies raised against A, B or RhD antigens
2) The sample is visually checked for agglutination (blood cells sticking together)
Agglutination indicates the presence of antigens in the blood sample.
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)
What is flow cytometry?
Technology used to analyse the proteins on cells that are in suspension.
It can determine:
- whether or not a cell expresses a target protein
- the amount of expression of a target protein
- Cell size, density and identity
It often involves the use of commercially produced antibodies that are conjugated to a fluorochrome.
Fluorochromes absorb light of a certain wavelength and emit light of a certain wavelength.
HOW IT WORKS
1) Fluorochrome-conjugated antibodies that are specific for the target protein are added to the cells.
2) The cells are passed through lasers that excite the fluorochrome (eg. blue laser excites FITC, which then emits green light)
3) The light emitted from the excited fluorochrome is detected and plotted on a graph.
The amount of light emitted is proportional to the amount of antibody bound to the protein, which is proportional to the amount of protein expressed by the cell.
CLINICAL APPLICATIONS
DIAGNOSTICS:
- diagnosis of haematological malignancies
- CD4 T-cell counts in HIV - HIV destroys white cells so its important to know how many you have to know the progression of the disease
e.g B cell lymphoma patients have reduced B cell and granulocyte counts/increased B cell clonality
RESEARCH:
- identification and analysis of immune cells in the context of infection and cancer immunotherapy
What is confocal microscopy?
Similar to flow cytometry, although there are some key differences:
- the cells to be analysed are not in suspension
- it’s 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 being plotted graphically
- confocal microscopy has the advantage of visualising where the protein is on the cell
Its applications are:
- mainly research
- indentification and analysis of cells within tissues
- co-localisation of different antigens
What is IHC?
IHC stands for ImmunoHistoChemistry. It’s used to show the distribution and localisation of antigens in tissue sections using visualisation of antibody-antigen interactions through chromogenic detection. IHC is useful since you can see where, in organs, certain things can be targeted to
METHOD
1) Thin sections of the tissue are cut.
2) Primary antibodies that recognise the target protein are added to the tissue.
3) The antibody-antigen interaction is visualised using chromogenic detection.
4) A secondary antibody specific for the primary antibody and conjugated to horseradish peroxidase (HRP) is added.
5) HRP catalyses the conversion of the chromogen 3,3-diaminobenzidine (DAB) substrate to produce a brown precipitate at the location of the protein.
6) The brown precipitate is then visualised by a light microscope.
WHEN IT IS USED
IHC is used routinely in the diagnosis of cancer.
Cancer can be caused by mutations in tumour supressor or tumour promoter genes (eg. BRAF is a gene that encodes the B-Raf protein that promotes cell division).
IHC is used to stain B-Raf protein in tissue sections of cancer patients in order to check for eligibility for treatment with B-Raf inhibitors.
CONTEXT:
- 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
What is ELISA?
Stands for Enzyme-Linked ImmunoSorbant Assay. It quantifies the amount of a protein or antibody in liquid samples such as sera or tissue culture supernatants.
There are four different types:
- direct
- indirect
- sandwich
- competitive
USES:
Direct ELISA - subsrate + primary antibody conjugate
Indirect ELISA - substrate + secondary antibody conjugate
Sandwich ELISA - substrate + capture antibody
Competitive ELISA - substrate + inhibitor antigen
APPLICATIONS
- antibody titres in patient serum (eg. viral infections such as HIV and Hepatitis B, as well as bacterial infections such as Mycobacterium tubercolosis (TB))
- Detection of bacterial toxins in food such as Escherichia coli O157:H7
- the home pregnancy test uses the principles of ELISA to measure human chorionic supernatants hormone (HCG) in urine
- in research, it is used to quantify cytokines/chemokines/growth factors in tissue culture supernatants
SANDWICH ELISA
1) add capture antibody
2) add the sample and wash
3) add the secondary with detection antibody (HRP)
4) add the substrate (TMB - chromogenic substrate)
5) determine the optical density using a spectrophotometer
What is Western Blotting?
Technique used to detect proteins in a tissue homogenate.
A Southern Blot (Edwin Southern) detects DNA
A Northern blot detects RNA
It involves four steps:
1) sample preparation
2) electrophoresis
3) transfer to membrane - Fractionated proteins are transferred onto a membrane
4) stain for protein of interest
METHOD
1) The sample is prepared by denaturing and lysing.
2) It is then loaded onto a gel for electrophoresis, where the proteins are separated based on size.
3) Then, it is transferred to a membrane.
4) We then stain for our protein of interest by incubating the membrane with a primary antibody specific for our target protein.
5) We then incubate the membrane with an HRP-conjugated secondary antibody specific for the primary antibody.
6) A chemiluminescent HRP substrate is added to the membrane.
7) The membrane is exposed to X-ray film that ‘bleaches’ when exposed to light.
APPLICATIONS
DIAGNOSTIC USE:
- Antibody detection of specific sized proteins from viral infections or parasites
- viral infections (eg. HIV)
- bacterial infections (eg. spirochete Borrelia burgdorferi that causes Lyme disease)
- used to detect the presence of antibodies against a pathogen in patient serum
- The pathogen is lysed and its proteins are seperated on a gel. The proteins are transferred to a membrane. the membrane is mixed with petient serum which may or may not contain antibodies. Secondary antibodies are added, etc.
RESEARCH:
- cell signaling proteins
- mechanism of action for cancer drugs
