Immunology in the clinic and research lab Flashcards
Selection of Hybridomas - explain
Selection of Hybridomas
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
Hybridomas - describe storage
Hybridomas can be stored indefinitely and grown to produce monoclonal antibody when required
What allows ABs to be engineered for different applications
Antibody genes can be cloned from the hybridomas which allows antibodies to be engineered for different applications
Anti-isotypic antibodies - define
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 - define
immuno
uses antibody-antigen interaction (one of which is “labelled” or “tagged” to allow its detection)
assays
measures (amount, concentration) of antibody or antigen
very sensitive and specific
hence, widely used in research and analytical labs
Immunoassays: the label - describe
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 - function of in/direct vs sandwich
Direct/Indirect
Often used to quantify an antibody
Sandwich (Capture)
Often used to quantify an antigen
The concentration of analyte (antibody or antigen) in the sample can be calculated by
Using these assays, the concentration of analyte (antibody or antigen) in the sample can be calculated by comparison to analyte standards of known concentration
Direct ELISA - describe process
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
Direct ELISA - list uses
uses
- screen hybridoma supernatants
- detect exposure to infectious agent
Indirect ELISA - describe 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
Indirect ELISA - explain how the sensitivity of the test is increased
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
Sandwich (Capture) ELISA - describe process
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
Immunoassays: Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and Western Blotting - uses
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
What is used alongside ELISA
SDS PAGE/Western blotting often used alongside ELISA
WB - describe measurement of [protein]
In WB, protein concentration can be measured by comparing intensity of band we are detecting to band from a protein standard of known concentration
ELISA - steps if protein is degraded and why
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
Antibody-antigen interaction: Flow cytometry and Fluorescence-activated cell sorter (FACS) analysis - describe process
Individual cells within a mixed population are tagged by treatment with monoclonal antibodies which bind to surface molecules and are labelled with fluorescent dyes
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
Types of sample that are analysed by biomedical scientists
Blood serum Blood cells Urine Synovial fluid Saliva Mucus Cerebrospinal fluid
Types of disease
Transplant compatibility Immunodeficiency Autoimmunity Allergy Malignancy
MHC alleles of donor and recipient are identified by
MHC alleles of donor and recipient are identified by Polymerase Chain Reaction
X-linked agammaglobulinemia (XLA). X-linked disorder - define
X-linked agammaglobulinemia (XLA). X-linked disorder- inability to generate mature B cells. B cells have CD19 surface protein which is a co-receptor for the B cell receptor
Flow cytometry in the clinic - monitoring HIV infection
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
Tracking T cell responses – fluorescent MHC complexes for antigen-specific T lymphocytes - describe process
MHC-peptide complexes made which bind specifically to the T cell receptor of appropriate MHC-peptide specific T cells. A fluorochrome e.g. phycoerythrin added and visualisation is by flow cytometry
By creating MHC complexes loaded with HIV antigen we can calculate for example what proportion of CD 8+ T cells will bind to the antigen
Neutrophil deficiencies - explain generation of pus
Neutrophils are found in acutely inflamed tissue
Ingest pathogens and kill using reactive oxygen species
Rapidly die after phagocytosis which generates pus
Deficiency in neutrophil numbers - effect
Deficiency in neutrophil numbers – neutropenia – high rate of infection
Deficiency in phagocyte function - effect
Deficiency in phagocyte function; chronic granulomatous disease – patients cannot form reactive oxygen species, succumb to bacterial and fungal infections
Measuring neutrophil function: Neutrophil Oxidative burst assay
This test is based on the principle that nonfluorescent DHR (dihydrorhodamine) 123 when phagocytosed by normal activated neutrophils (after stimulation with PMA – phorbol myristate acetate) can be oxidized by reactive oxygen species (ROS),
produced during the activated neutrophil respiratory oxidative burst, to rhodamine 123, a green fluorescent compound, which can be detected by flow cytometry.
Quantitation of antibodies: Nephelometry - describe
Electrophoresis
Nephelometry:an automated and rapid method used to measure serum immunoglobulin levels; it relies on the light-scattering properties of antigen-antibody complexes
Quantitation of antibodies: Nephelometry - use
Nephelometry often used to study the amount of antibody from different classes present in serum e.g. IgG, IgA etc
Skin Prick Test: - describe
Skin Prick Test: In allergic individual, 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
RAST (RadioAllergoSorbent Test) - describe
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
Immunofluorescence: - describe process
Immunofluorescence: serum added to human cell line. Then probed with fluorochrome-labelled anti-immunoglobulin antibody. Visualised by fluorescence microscopy
Monoclonal antibodies can have their effects either by what
Monoclonal antibodies can have their effects either by binding and blocking a process or by mediating immune responses such as initiation of complement or antibody-dependent cell mediated cytotoxicity (ADCC)
What do monoclonal antibodies bind to
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
