40. Immunology Clinic and Research Lab Flashcards
What are the different regions on an antibody molecule (IgG)
•Fab region
- Has complementarity determining regions (CDRs)
- Antibody repertoire
- Affinity
- Avidity
•Fc region
- Antibody dependent cell-mediated cytotoxicity (ADCC)
- Antibody dependent cellular phagocytosis (ADCP)
- Complement dependent cytotoxicity (CDC)
Describe the polyclonal antibody response
- mouse injected with antigen w/different epitopes
- antibody response ~ polyclonal antibodies produced
- binding of epitopes to the B cells will cause them to be activated and proliferate ~ they secrete the complimentary antibodies.
- polyclonal antibodies
Describe the production of monoclonal antibodies by hybridoma culture
1) Antigens injected in mouse and harvest B cells producing the complementary antibody
2) Fuse these with myeloma cells ~ called hybridomas
3) Hypoxanthine-aminopterin-thymidine (HAT) selection gets rid of the unfused cells
4) Propagate desired clones
5) Grow the cells in mass culture
6) Harvest the monoclonal antibodies
Describe the selection in the HAT medium
In HAT medium, myeloma cells die as they cannot make nucleotides due to lack of HGPRT gene. B cells die as they have a short life span. Only hybridomas grow and proliferate
anti-isotypic antibodies
Polyclonal or monoclonal antibodies can be produced which bind to Fc regions of particular antibody classes e.g. to IgG’s, IgA’s etc. These are called anti-isotypic antibodies
Immunoassays label
•originally radioactive
- radioimmunoassay (RIA)
•commonly now enzyme e.g. horseradish peroxidase or alkaline phosphatase -usually detected by coloured product (colorimetric)
- enzyme-linked immunosorbent assay (ELISA)
• other alternatives are luminescent
Solid phase immunoassays: e.g. ELISA
Describe the function of Direct/indirect ELISA, and sandwich
DIRECT/INDIRECT
- Often used to quantify an antibody
SANDWICH (CAPTURE)
- Often used to quantify an antigen
~ Using these assays, the concentration of analyte (antibody or antigen) in the sample can be calculated by comparison to analyte standards of known concentration
Expand on direct ELISA
- antigen immobilised on solid support
- test antibody solution covalently linked to enzyme (e.g. Horseradish peroxidase or alkaline phosphatase for colorimetric ELISA) added
- Enzyme substrate added, coloured product produced which can be measured by absorbance
USES
•screen hybridoma supernatants
•detect exposure to infectious agent
Expand on 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
Secondary antibody is often polyclonal and so may bind to different epitopes on a primary antibody. This allows multiple secondary antibodies to bind to the same primary antibody thereby amplifying the signal and increasing the sensitivity of the test
Expand on 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
Measuring Cytokine secretion - Elispot
- Cytokine-specific antibodies are bound to the surface of a plastic well
- Activated T cells are added to the well.
- Cytokine secreted by some activated T cells is captured by the bound antibody
- Second cytokine-sepcific antibody is coupled to enzymes that gives rise to a spot of insoluble coloured precipitate
Expand on Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and Western Blotting
- You start off taking the sample with the protein you are trying to detect
- You take the sample and you boil it with sodium dodecyl sulphate
- This will bind to the protein and give the protein a net negative charge
- You then run the protein on a polyacrylamide gel – runs according to size
- You then take the gel and you blot it onto Nitrocellulose (which is like paper)
- You then probe the Nitroccellulose with an antibody that is linked to an enzyme that gives a coloured product
- When you add the substrate, you will see a band form on the nitrocellulose
1) Can be used to detect antigens or antibodies
2) Used to measure size of the protein being analysed
3) Can be used to calculate protein concentration
4) May show if protein has been degraded
- used alongside ELISA
- 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
Expand on antibody coated magnetic beads
- lypmphocytes are mixed with antibodies coupled to magnettic beads/particles and poured over an iron wool
- magnetic field applied, coupled cells stick to the iron wool and the unlabelled cells are washed out
- magnetic field is removed and releases the coupled cells
Expand on Fluorescence-activated cell sorter (FACS) analysis
Mixed cells are then forced through a
nozzle to form stream of single cells
Individual cells pass through a laser
beam which scatters light and causes dye to fluoresce and
provides information on bound antibody and cell surface protein
MHC typing for transplant compatibility
• MHC alleles of donor and recipient are identified by Polymerase Chain Reaction
