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
What are the types of samples that are analysed
- Blood serum
- Blood cells
- Urine
- Synovial fluid
- Saliva
- Mucus
- Cerebrospinal fluid
What types of diseases are immunoassays used in
- Transplant compatibility
- Immunodeficiency
- Autoimmunity
- Allergy
- Malignancy
How can we avoid an unwanted immune response in a transplant
- Histocompatibility – genetic differences between
individuals are detected by the immune system,
leads to rejection of non-self - Major histocompatibility proteins (MHC) are major
players in transplant rejection - Best transplant results when donor and recipient
MHC are as similar as possible
How is MHC type matching carried out
MHC alleles of donor and recipient are identified by Polymerase Chain Reaction
How do we use immunoassays in immunodeficiencies to quantify cell populations
- Use of flow cytometry to identify what markers cells express
How can flow cytometry be used in HIV
- Lymphocyte subset estimations are performed using
monoclonal antibodies to: CD3, CD4 and CD8 on whole
blood and analysed by flow cytometry - The percentages of cells in each subset is determined using
a FACS machine - The results are reported as percentages and absolute
counts
How do we use immunoassays in immunodeficiencies to analyse cell functions
- Measure function neutrophils by labelling them with DHR
- Activated neutrophils will be fluorescent and can be measure by flow cytometry
How do we use immunoassays in immunodeficiencies to analyse antibody levels
- Use nephelometry
- Use light scattering to quantify antibody levels
How do we use immunoassays in allergies
- Use antigen skin prick test and observe reaction on skin
- Use of RAST test to quantify IgE in serum
How can we use immunoassay sin autoimmunity
- Grow cells in vitro
- Incubated with serum from SLE patients
- Then probed with fluorochrome-labelled anti-immunoglobulin
- Visualised by fluorescence microscopy
Describe the uses of polyclonal antibodies in therapy
- Administering IV immunglobulins
- Uses in antibody deficiencies
- Use in rabies
Describe the uses of monoclonal antibodies in therapy
- licensed to treat disease: cancer, chronic inflammatory
diseases, transplantation, infectious diseases and cardiovascular medicine - initiation of complement or antibody-dependent cell
mediated cytotoxicity - Block toxins or labelled for target of radiotherapy
