Immunology in the Clinic and Research Lab Flashcards
What is the general structure of an antibody?
β Two heavy chains and two light chains held together by disulfide bridges
What two parts can an antibody be divided into?
β Fab region
β Fc region
What does the Fab region bind to?
β Binds to antigens
β light chain binds to antigen epitopes via the CDR
What is an antibody repertoire?
β all the possible numbers of antibody binding sites
What is antibody affinity?
β the strength of a single interaction between the antibody and epitope
What is antibody avidity?
β sum of different affinities
What are the 5 functions of the Fc region?
β INteracts with the immune system - ADCC
β mediates antibody dependent cell mediated cytotoxicity - ADCC
β antibody dependent cellular phagocytosis - ADCP
β complement dependent cytotoxicity
β pharmacokinetics/half life
Describe how a polyclonal response occurs if you inject a mouse with an antigen?
β the antigen has many epitopes on it
β after a week an antibody response is generated to the antigen
β there are B cells that will produce antibodies to different epitopes of the antigen (polyclonal response)
β The B cells proliferate and form clones of themselves and secrete antibodies which are all the same specificity as the original B cell
Who invented monoclonal antibodies?
β Milstein and Kohler
Describe how monoclonal antibodies are made in a lab?
1) You take the antigen that you want antibodies against and inject it into a mouse
2) After a week or two weeks you harvest the B cells which make the antibody
3) The B cells are taken and are fused using polyethylene glycol with a cell-culture line of myeloma cells
4) The myeloma cells also lack the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene
5) After the fusion has occurred there is a mixture of cells : unfused B cells and myeloma cells, and fused cells called hybridomas
6) The next stage is trying to select for the hybridomas and remove the unfused cells
7) This is done using a selection called HAT (hypoxanthine-aminopterin-thymidine) selection
8) In HAT medium myeloma cells die as they cannot make nucleotides due to lack of HGPRT gene.
9) B have the gene but cells die as they have a short life span, only hybridomas grow and proliferate
10) Then there is a mixture of hybridoma cells which are diluted to individual cells
11) The cells are cultured individually they proliferate to form a clone of cells identical to the original parent
What are myeloma cells?
β immortal cells derived from a B cell tumor that do not produce antibodies themselves
What are monoclonal antibodies?
β antibodies that bind specifically to one epitope
How long can hybridomas be stored for?
β indefinitely
What are isotypic antibodies?
β polyclonal or monoclonal antibodies that bind to Fc regions of particular antibody classes e.g IgG or IgA
What is an assay?
β measures amount or concentration of antibody or antigen
What 2 enzymes are used as labels in assays?
β Horseradish peroxidase
β alkaline phosphatase
What is an example of a solid phase immunoassay?
β ELISA test
What are the three types of ELISA test?
β Direct
β Indirect
β Sandwich
What are direct/indirect ELISA tests used for?
β to quantify an antibody
What are sandwich ELISA tests used for?
β to quantify an antigen
How does a direct ELISA test work?
β The antigen is immobilised on a solid support
β the test antibody solution covalently linked to enzymes is added
β enzyme substrate is added and a colored product is produced which can be measured by absorbance
What are the two uses of the direct ELISA test?
β screen hybridoma supernatants
β detect exposure to an infectious agent
How does an indirect ELISA test work?
β Antigen immobilised on a solid suport
β primary antibody which binds to the antigen is added
β secondary antibody covalently attached to an enzyme is subsequently added
β the secondary antibody binds to the Fc region of the primary antibody
β the enzyme substrate is added and color is measured by absorbance
Why is an indirect ELISA used?
β the second antibody is often polyclonal so it can bind to different epitopes on a primary antibody
β this allows multiple secondary antibodies to bind to the same primary antibody
β this amplifies the signal and increases the sensitivity of the test
What is the sandwich ELISA used for?
β concentrating the antigen when the antigen is present in a low concentration
What is needed for a sandwich ELISA?
β Two antibodies reacting with different epitopes
How does a sandwich ELISA test work?
β One antibody is immobilised on a solid support
β the test antigen solution is added, incubated and non-bound antigen is removed by washing
β the bound antigen detected by incubation with the other antibody which has been labelled
β non bound removed by washing
What is the name of an immunoassay done to measure cytokine secretion?
β Elispot
How does an Elispot work?
β There are antibodies specific to the cytokine you are trying to detect
β they are immobilised at the bottom of an ELISA plate
β you add activated T cells to the plate
β the T cells will secrete the cytokines
β the cytokine will bind to the antibody
β a second antibody conjugated to an enzyme is added to detect the colored product
What are the 4 things a western blot and an SDS PAGE can be used for?
β Can be used to detect antigens or antibodies
β used to measure the size of the protein being analysed
β can be used to calculate the protein concentration
β may show if a protein has been degraded
How does a western blot work?
β you take the sample with the protein you are trying to detect and boil it with sodium sulfate, this will bind to the proteins and give it a net negative charge
β then you run the protein in gel
β the protein will run according to its size
β the proteins from the gel are blotted onto nitrocellulose
β you can probe the nitrocellulose with an antibody linked to an enzyme that gives a colored product
β when you add the substrate you can see a band that forms on the nitrocellulose
What is SDS PAGE/western blotting used alongside?
β ELISA
How can protein concentration be measured in western blotting?
β Compare the intensity of the band you are detecting to a band from a protein standard of a known concentration
If a protein is degraded why is it more useful to use a western blot to calculate protein concentration?
β Some of the degradation fragments may contribute a signal in ELISA if both the coating and detecting antibody are able to bind to them
Describe how you can use antibody-antigen interaction for purification of immune cell subsets?
1) The antibodies are coated with magnetic beads
2) The antibodies will bind specifically to certain proteins on the surface of the immune cell that you are trying to isolate
3) Once it is bound you can put the cells with the antibodies into a machine with an iron wall mesh
4) You can apply a magnetic field and only the cells which have the antibody coated in magnetic particles will bind to the iron mesh and the other cells with be washed out
5) You can remove the magnetic field and elute the cells
6) The cells will be the type you wanted to isolate
Describe how you can use flow-cytometry and antibody-antigen interaction to sort cells ?
1) Antibodies bind to a particular cell that binds to a protein on its surface
2) The antibodies are labelled with a fluorescent dye
3) Once there is a mixture of cells you put them into the flow cytometer
4) They are passed through a very thin nozzle to form a single line of cells
5) As that happens a laser beam is shone through as the cells go through the nozzle
6) Individual cells pass through the laser beam which scatters light and causes the dye to fluoresce and provides information on bound antibody and cell surface protein
What leads to rejection of non-self?
β genetic differences between individuals
What does MHC class I bind?
β fragments of intracellular proteins
What does MHC class II bind?
β Fragments of proteins which have been taken up by endocytosis
Where is the MHC gene located?
β chromosome 6
How are MHC alleles of donor and recipient identified?
β PCR
How do you separate a control and X linked agammaglobulinaemia cells with flow cytometry?
β B cells have a surface protein called CD19
β tag the CD19 with an antibody
β put it through a flow cytometer
β in a normal individual you will have cells that express CD3 (T cells) and CD19
β X linked only has CD3 cells
How are lymphocyte subset estimations done with flow cytometry?
β monoclonal antibodies to CD3, CD4 and CD8
β the percentage of cells in each subset is determined using flow cytometry
How do you track T cell responses using flow cytometry?
1) MHC specific complexes made which bind specifically to the TCR of t clells
2) At the C terminal end biotin has been added which binds strongly to streptavidin linked to a fluorochrome
3) A fluorochrome e.g phycoerythrin is added and visualisation is done by flow cytometry
4) Interaction between TCR and MHC is usually quite weak, having a tetramer makes binding more likely
5) By creating MHC complexes loaded with HIV antigen we can calculate what proportion of CD8 + T cells will bind to the antigen
Where are neutrophils found?
β acutely inflamed tissue
What do neutrophils do?
β Ingest pathogens and kill using reactive oxygen species
What generates pus?
β Neutrophils rapidly die after phagocytosis
What is neutropenia?
β a deficiency in neutrophil numbers
What is the outcome of a deficiency in phagocyte function?
β chronic granulomatous disease
β patients cannot form ROS and succumb to bacterial and fungal infections
How do you measure neutrophil function?
β Neutrophil oxidative burst assay
How does the neutrophil oxidative burst assay work?
β Non fluorescent dihydrorhodamine when phagocytosed by normal activated neutrophils (after stimulation with PMA - phorbol myristate acetate) can be oxidised by ROS
β ROS oxidises DHR to rhodamine 123, a green fluorescent compound
When are ROS produced?
β during the activated neutrophil respiratory oxidative burst
What is nephelometry?
β An automated rapid method used to measure serum immunoglobulin levels
What does nephelometry rely on?
β light scattering properties of antigen-antibody complexes
How does nephelometry work if you want to study IgG?
β You take some serum and add anti IgG (anti-isoantibody)
β they form complexes and light is shone through the serum
β the complexes diffract the light and the amount of diffraction is measured and it will correspond to the number of the complexes
What antibody is predominant in allergic responses?
β IgE
What causes reddening and swelling of the skin during the allergic response?
β IgE binds to an allergen and via the Fc region of the antibody binds to receptors on mast cells
β this causes mast cells to degranulate causing the release of mediators (histamine) which causes redness and swelling
How does the skin prick test work?
β prick the skin with potential allergens, if they are allergic there will be a bump
How is RAST done?
β the suspended allergen is bound to an insoluble material and the patients serum is added
β if the serum contains antibodies to the allergen the antibodies will bind to it
β radio-labelled anti human IgE antibody is added and it binds to the IgE antibodies already bound to the insoluble material
β the amount of radioactivity is proportional to the serum IgE for the allergen
What does RAST stand for?
β radioallergosorbent test
What two tests are done for SLE?
β ELISA (quantitative)
β immunofluorescence (qualitative)
How is immunofluorescence done for SLE patients?
β serum is added to a human cell line
β it is probed with fluorochrome labelled anti-immunoglobulin antibody
β visualised by fluorescence microscopy
What % of SLE patients are anti-nuclear antibody positive?
β 98%
What are IV immunoglobulins?
β a blood product purified from the serum of between 10,000-15,000 people
What are IV immunoglobulins used to treat?
β patients with antibody deficiencies
How much IV immunoglobulin is used for treatment?
β 200-400mg/kg/3 weeks
What is a high dose of IV immunoglobulin and what is this used for?
β 2g/kg/4 weeks
β immunomodulatory agens in immune and inflammatory disorders
What is given if a person contracts rabies?
β Polyclonal antibodies isolated from the serum of individuals who have been immunised with the rabies vaccine are injected into the wound site
How do monoclonal antibodies exert effects in treatment?
β binding and blocking a process
βmediating immune responses such as the initation of complement or antibody cell mediated cytotoxicity
How are monoclonal antibodies used to kill cancer cells?
β Toxins or radionuclides can be joined to monoclonal antibodies
β the antibody binds to the cancer cell which is then killed by toxin or radioactivity
