Antibodies - Uses
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List the uses of antibodies
Immunostaining Flow Cytometry Immunoassays Immunoprecipitation Western blotting
Why is immunostaining done? 3
To detect cell and tumour proteins To identify micro-organisms - detect the presence of some diseases To investigate cell phenotype
How is immunostaining carried out?
Antibody labelled with fluorescent dye or enzyme which creates coloured deposit at site of antigen
In what lab is immunostaining carried out?
Histology laboratory
How can immunostaining be used for cell and tumour identification?
It can tell us where the primary tumour is from It can tell us what tumour subtype it is
Explain in your own words what immunostaining is. 3
An antibody is used to stain a sample The antibody is labelled with fluorescent dye or an enzyme A coloured deposit is created at the site of the antigen - a specific protein epitope
What is flow cytometry
A method of cell sorting and quantitation
Explain how flow cytometry works in your own words.
Similar to water going down a sink As it spins the cells are in a line that passes by a laser A flash will occur when the selected antibody and attached cell pass by the laser The number of the cell type is counted as each one passes by the laser The machine will also separate the chosen cell type from the sample
How is the cell separated from the sample in flow cytometry? 2
As the chosen cell passes through the device a charge is applied to the cell The machine then pulls the chosen cell away from the sample
Give an example of a type of cell used in flow cytometry?
Stem cells
What antibody is used to count stem cells?
CD-34
What is an immunoassay also called?
Ligand-binding assay
What is the basis of immunoassays?
A broad range of techniques whose common feature is that measurement is based on specific recognition of the analyte by an antibody or other macromolecule
What are the four main immunoassay methods?
Radio-labelled RIA Enzyme-labelled EIA Chemiluminescence Fluorescence- labelled
What is a radio-labelled RIA? 2
An antibody is labelled with radioactive isotope The radioactivity is measured
Why is a radio-labelled RIA rarely used?
It is dangerous and needs a special lab
What is an enzyme-labelled EIA? 3
ELISA An antibody is labelled with an enzyme Colour reaction is measured spectrophotometrically
Why is an ELISA not often used?
Enzymes are too valuable to be used
What is chemiluminescence?
An antibody is labelled with chemiluminescent dye
What is fluorescence labelled? 2
An antibody is labelled with fluorescent dye The fluorescence is measured
What are immunoassays used for? 2
Measuring blood/fluid constituents Detection of specific proteins in fluid samples
What is immunoprecipitation used for?
The detection of proteins and bacteria
How is immunoprecipitation used to detect bacteria?
Bacteria are detected using antigen-antibody reactions
Explain in your own words how immunoprecipitation works - using Syphilis as an example (6)
Latex beads are coated in anti syphilis antibody Syphilis reacts with beads This causes beads to clump together Drop of latex beads and drop of patients plasma Drops will disperse and stain the gel If a line appears between antigen and antibody then the test is positive