Immunology in the Clinic and research lab Flashcards
Define “immunology” in immunoassay
uses antibody-antigen interaction (one of which is “labelled” or “tagged” to allow its detection)
Define “assays” in immunoassay
measures (amount, concentration) of antibody or antigen
Describe how polyclonal antibodies are produced
- Immunise animals with a particular antigen
- Extract serum from that animal
- Purify antibodies present in serum
- After immunising the animal will produce specific B cells that produces specific antibodies
How are monoclonal antibodies produced
- Immunise mouse with antigen
- Harvest B cells from mouse
- Fuse B cells with myeloma cells in polyethylene glycol
- Select hybrid cells from the population by culturing them on HAT
- Propagate the desired colonies and dilute to one cell per well
What are the basic principles of labels in immunoassays
- Originally radioactive
- Commonly now enzymes such as in ELISA
- Others are luminescent
What are the different formats of solid phase immunoassays
- Direct/Indirect - used to quantify an antibody
- Sandwich (capture) - Used to quantify an antigen
What are the basic principles of a Direct ELISA
- antigen immobilised on solid support
- test antibody solution covalently linked to enzyme added
- Enzyme substrate added, coloured product produced
which can be measured by absorbance
What are the uses of direct ELISA
- Screen hybridoma supernatants
- Detect exposure to infectious agents
What are the basic principles of indirect ELISA
- antigen immobilised on solid support
- primary antibody which binds to antigen is then added
- Secondary antibody covalently attached to enzyme is subsequently added. Secondary antibody binds to Fc region of
primary antibody - Enzyme substrate added, colour measured by absorbance
What are the basic principles of Sandwich (capture) ELISA
- Antigens may be present in low concentration. Because antibodies have high affinity for antigen this technique can concentrate the antigen
- need two antibodies reacting with different epitopes on the antigen
- one antibody immobilised on solid support
- test antigen solution added, incubated and non-bound removed by washing
- bound antigen detected by incubation with the other antibody, which has been labelled, and non-bound removed by washing
Describe the Elispot assay
- Have a solid support which is coated with antibody
- Add T cells to the well
- If the cell releases cytokines they bind to antibody
- A second antibody is bound to cytokine to form a sandwich
- The cells are then eliminated
- Spots are observed in the wells
What can western blotting be used for
- Can be used to detect antigens or antibodies
- Used to measure the size of the protein being analysed
- Can be used to calculate protein concentration
- May show if protein has been degraded
- Similar to gel electrophoresis
How can we use a combination of techniques for analysis
- In WB, protein concentration can be measured by comparing intensity of band
we are detecting to band from a protein standard of known concentration - If protein is degraded it may be more useful to use WB to calculate protein
concentration, as some of degradation fragments may contribute to signal in
ELISA if both coating and detecting antibody are able to bind to them
Describe how we can purify immune cells with antibody-coated magnetic beads
- Heterogenous population of lymphocytes are mixed with antibodies coupled to paramagnetic particles or beads and poured over an iron wool mesh
- When a magnetic field is applied the coupled cells stick to the iron wool, and unlabelled cells wash out
-The magnetic field id removed releasing the coupled cells
Describe ether use of lateral flow tests
Different LFT formats exist. Are used for many applications. Can be used to detect different types of molecules including antibodies
