Immunological Techniques In Diagnostics And Research Flashcards
What is a hybridoma?
B cells and tumour cells fused together
What do the hybridomas produce?
Antibodies against the target protein
What is a monoclonal antibody?
Only has one binding site for one specific shape on one specific antigen
What is the blood sample mixed with when they’re trying to work out a blood type?
Antibodies raised against A,B or RhD
What happens to the RBCs when they’re mixed with antibodies in blood typing?
Agglutination
What happens in blood typing if the samples are all positive or negative?
You’ve done it wrong
What is flow cytometry?
Technology used to analyse the proteins on cells that are in suspension
What can flow cytometry tell you (4)?
- cell size and density
- if a cell expresses a target protein
- The amount of expression of a target protein
- The cells identity
What does flow cytometry often involve the use of?
Commercially produced antibodies that are then conjugated to fluorochromes
What is a fluorochrome?
A fluorescent molecule that absorbs light of a certain wavelength and in turn emits light of a certain wavelength
What is detected in flow cytometry?
The amount of light emitted
How is flow cytometry used in research(2)?
Identification and analysis of immune cells
Cell sorting
How is flow cytometry used in diagnostics(2)?
CD4 T cell counts in HIV
Diagnostics of haematological malignancies
What happens to the immune cells in B cell lymphoma?
Reduced T cell and granulocytes and increased B cell clonality
Why do you need to use flow cytometry in HIV?
HIV destroys white cells so its important to know how many you have to know the progression of the disease
What are 3 applications of confocal microscopy?
Mainly research
Identification and analysis of cells within tissues
Co-localisation of different antigens
Name the four differences between confocal microscopy and flow cytometry
In confocal microscopy:
- 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 (not plotted graphically)
- has the advantage of visualising where the protein is in a cell
What does IHC stand for?
Immunohistochemistry
What is IHC used for?
To show the distribution and localisation of antigens in tissue sections using antibody-antigen interactions
What does a brown precipitate mean in IHC?
The protein is present
Why is IHC helpful?
You can see where, in organs, certain things can be targeted to
How is IHC used in cancer diagnosis?
Used to stain B-Raf protein in tissue sections selecting patients eligible for B-Raf inhibitor therapy
What is visualised using chromogenic detection in IHC?
The antibody-antigen interaction
What is the secondary antibody conjugated to in IHC?
Horseradish peroxidase (HRP)
What does the horseradish peroxidase do in IHC?
Catalyses the conversion of the chromagen 3,3-diaminobenzidine substrate to produce a brown precipitate
What does ELISA stand for?
Enzyme-linked immunoabsorbant assay
What does the ELISA test do?
Quantifies the amount of a protein or antibody in liquid samples such as sera or tissue culture supernatants
What are the 4 applications of ELISA tests?
Antibody titres sera (HIV and HepB)
Bacterial toxins in food
Home pregnancy testing
Research quantification of cytokines/ chemokines/ growth factors in tissue culture supernatants
What are the four types of ELISA?
Direct
Indirect
Sandwich
Competitive
Give the 6 steps in the sandwich ELISA
Add capture antibody Add sample Wash Add detection antibody Add substrate Determine optical density
What does western blotting do?
Detects proteins
What are the 2 applications of western blotting?
Antibody detection of specific sized proteins from viral infections or parasites
Cell signalling proteins
What are the four steps of a western blot?
Sample prep
Electrophoresis
Transfer to membrane
Stain for protein of interest
What happens in the sample prep step of a western blot?
Cells are lysed and proteins denatured
What happens in the electrophoresis step of a western blot?
Lysates are loaded onto a gel and proteins are separated based on size
What happens in the transfer to membrane step of a western blot?
Fractionated proteins are transferred onto a membrane
How are the proteins transferred onto a membrane in a western blot?
Put filter paper on top and pass electricity through it onto the paper
What happens in the staining step of a western blot (4 steps)?
- the membrane is incubated with a primary antibody specific for the target protein
- the membrane is incubated with a HRP conjugated secondary antibody for the primary antibody
- a chemiluminescent HRP substrate is added to the membrane
- the membrane is expose to an X-ray film that bleaches when exposed to light
