Antibodies - Uses Flashcards
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
What are the two different ways of carrying out immunoprecipitation?
(2)
Using one drop of the latex beads coated in anti syphilis antibody and one drop of the patients plasma
One drop with antigen and one drop with antibody
Explain in your own words how western-blotting works.
6
Electrophoresis - plastic is put on gel
The gel is dried out
Bands stick to plastic
Stain with antibody which has a radiolabel in it
Photograph it
Antigen bands develop on film
