ImmunoRes Flashcards
Describe the production mAb
→inject mouse with antigen to produce Bcells
→cell culture myeloma line- lack HAT
→fuse myeloma and Bcells in polyethene glycol
→select hybrid cells using hypoxyanthine-aminopterin-thymidine(HAT)
→propagate desired clones
Why is HAT medium used to culture myeloma cells?
→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
How long can hybridomas be store for?
→indefinitely
What do immunoassays use?
→use polyclonal or monoclonal antibodies
What is the common enzyme used to label antibodies?
→horseradish peroxidase
→alkaline phosphatase
→usually colometric
What is an example of an immunoassay which uses horse radish peroxidase?
→ELISA
What are the two types of ELISA?
→direct/ indirect
→sandwich (capture)
What is direct/ indirect ELISA often used for?
→to quantify an antibody
What is sandwich ELISA often used for?
→quantify an antigen
What is direct ELISA mainly used for?
→screen hybridoma supernatants
→detect exposure to infectious agent
What is immobalised on the solid in indirect ELISA?
→antigen
Describe indirect ELISA process
→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 is a feature of secondary antibody in indirect ELISA and why is this a benefit?
→polyclonal and so may bind to different epitopes on a primary antibody
→increases sensitivity
Describe the process of sandwich ELISA
→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
What is Elispot?
→measuring cytokine secretion
What can SDS-PAGE and Western blotting be used for?
→1) Can be used to detect antigens or antibodies
→Used to measure size of the protein being analysed
→Can be used to calculate protein concentration
→May show if protein has been degraded
Which is method is more useful if protein has been degraded rather than ELISA?
→Western Blot
Which chromosome is is the HLA gene found on?
→6
How can antibodies to cell surface markers identified?
→flow cytometry
What test is used for tracking Tcell responses?
→fluorochrome e.g. phycoerythrin added and visualisation is by flow cytometry
What is disease involving neutrophil deficiency?
→chronic granulomatous disease (CGD)
How to measure neutrophil function?
→Oxidative burst assay
→green fluorescent compound, which can be detected by flow cytometry
What test is used to quantify antibodies?
→Nephelometry
→light-scattering properties of antigen-antibody complexes
Why does a skin prick test work?
→IgE binds to allergen and via the Fc region of the antibody binds to receptors on mast cells.
→This causes mast cells to degranulate causing the release of mediators (e.g histamine) which causes reddening and swelling of skin
What is a test for allergy diagnosis?
→RAST (RadioAllergoSorbent Test)
→The suspected allergen is bound to an insoluble material and the patient’s serum is added.
→If the serum contains antibodies to the allergen, those antibodies will bind to the allergen.
→Radiolabeled anti-human IgE antibody is added where it binds to those IgE antibodies already bound to the insoluble material.
→The amount of radioactivity is proportional to the serum IgE for the allergen
What are autoimmune diseases characterised by?
→autoantibodies to nuclear antigens eg DNA, RNA
How can SLE be diagnosed?
→Immunofluorescence
→serum added to human cell line
→Then probed with fluorochrome-labelled anti-immunoglobulin antibody.
→Visualised by fluorescence microscopy
How can antibodies be used for therapy?
→Intravenous immunoglobulin (IVIG
→eg rabies
How do mAbs exert their effects?
→binding and blocking a process
→mediating immune responses such as initiation of complement or antibody-dependent cell mediated cytotoxicity (ADCC)
→Molecules such as toxins or radionuclides can be joined to monoclonal antibodies: antibody binds to cancer cell which is then killed by toxin or radioactivity
