Immunological Techniques in Diagnostics & Research Flashcards
Describe the structure of antibodies
Membrane-bound (B cell receptor)
Secreted
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
How do we raise antibodies?(6)
Mice are immunised with the target protein
B cells are harvested & fused with tumour cells to form hybridoma
Tumour cells are drug sensitive but not b-cells
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
Monoclonal antibodies have one binding site for one antigen.
How does agglutination 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 are the 4 blood types?
A, B, AB & O
What is Rhesus factor?
(D protein or RhD) is also a protein on the surface of RBCs (positive or negative)
What is Flow cytometry?
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 on the Fc
How a flow cytometer works?(4)
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 light 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
Name some gated filters in flow cytometry
CD3+CD4+ and CD3+CD8+ T cells
What are the applications of flow cytometry?(2)
Diagnostics: CD4 T cell counts in HIV
Diagnosis of haematological malignancies
Research: Identification & analysis of immune cells
What is the role of confocal microscopy?
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
What is a difference between confocal and flow cytometry?
The cells to be analysed are not in suspension
What is an advantage of confocal microscopy?
visualising where the protein is on the cell
What are the applications of confocal microscopy?
Mainly research eg. Zebrafish
Identification & analyse cells within tissues
Co-localisation of different antigens
What is IHC? (3)
IHC stands for ImmunoHistoChemistry
Used to show the distribution & localization of antigens in tissue sections using antibody-antigen interactions.
Very reliable and can be used to localise antibodies
How does IHC work?(7)
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 chromogenic detection
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
You can see cells that are most likely there eg cancer cells and where they are made
Examples of IHC…
IHC is routinely used in the diagnosis of cancer
IHC is used to stain B-Raf protein in tissue sections from cancer patients and direct eligible for treatment with B-Raf inhibitors
What is a BRAF gene?
a gene that encodes B-Raf protein that promotes cell division
What are Vemurafenib and dabrafenib?
are inhibitors used to treat B-Raf-positive tumours
What is ELISA?
Enzyme-Linked ImmunoSorbant Assay
Quantifies the amount of a protein or antibody in liquid samples such as sera or tissue culture supernatants
Applications of ELISA…
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
Name the 4 types of ELISA
Direct
Indirect
Sandwich
Competitive
What does density of blue solution indicate in sandwich ELISA?
Amount of protein in a sample
What is the process Western blotting?
- Sample preparation
Cells are lysed & proteins denatured - Electrophoresis
Lysates are loaded onto a gel & proteins separated based on size - Transfer to membrane
Fractionated proteins are transferred onto a membrane - Stain for protein of interest
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
What are the applications of Western blotting?
Diagnostic use
Research: Cell signalling proteins. Mechanism of action for cancer drugs
How is Western blotting used as a diagnostic tool for pathogens?
Diagnostic use
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.
spirochete Borrelia burgdorferi that
causes Lyme disease
