Immunological Methods Flashcards
What is competitive binding immunoassay?
Antigen (Ag) present in samples to be assayed competes with a fixed amount of labelled Ag in the presence of a limiting quantity of antibody (Ab).
At equilibrium, free Ag is separated from Ab-bound Ag and the amount of labelled Ag present in the latter determined by scintillation (small flash of light) or Y-counting.
Name three immunoassays.
Competitive binding immunoassays.
Enzyme-linked immunoabsorbant assay (ELISA)
Enhanced immunoassay
Why is competitive binding immunoassay used?
For detection of steroids, peptide hormones in human/animal serum and plasma
Immunoglobulins
What are the benefits of CBI?
Very sensitive
Precise, accurate and economical (small amount of Ab is required)
What are the problems with CBI?
Difficult to automate
Time-consuming
Narrow range of concentration
Radiation controls
What is immunohistocytochemistry?
Determine the distribution of an antigen in situ.
Used to understand; cell structure, organisation, cell or tissue development.
Used for diagnosis of cancer.
What are the steps for IHC?
Ab must be conjugated with a fluorescent or enzyme
Sections of cells
Microscopy
The location of the label reveals the site of the Ab-Ag interaction.
Why use IHC for cancer detection?
It detects cancer in its earliest stages which increases survival, decrease in expensive therapy, invasive procedures, reduction in bed occupancy, lessening of the burden on the health care system.
What is flow cytometry?
Cells have been labelled with a fluorchrome-conjugated Ab.
Cells are introduced into a liquid gel and passed individually through the beam of a laser.
As each cell passes through it emits a flash of fluorescence and scattered light.
The signals are to give quantitative information on the intensity of fluorescence of each cell.
Why is forward angle light scatter used?
It determines cell size.
What is antibody microarrays?
Antibody microarrays contain hundreds of monoclonal antibodies immobilised onto a glass surface, allowing for the comparison of multiple proteins in two biological samples.
The antibody microarray is a complete analysis system for profiling protein expression in biological samples.
This new technology advances the current state of proteomics allowing researchers to compare the relative abundance of hundreds of proteins in a single experiment.
The antibody microarray is designed to be used as a screening tool for correlating proteins with a physiological or pathological process.
In one experiment you can profile protein changes associated with apoptosis, cancer, cell-cycle, neurological pathways and protein kinases.
What are the three antibody detection techniques?
1) direct technique (labelled primary antibody)
2) indirect technique (labelled secondary antibody)
3) biotin/avidin technique (using biotinylated secondary antibody and labelled streptavidin).
What is ELISA?
A sensitive immunoassay that uses an enzyme linked to an antibody or antigen as a marker for the detection of a specific protein, especially an antigen or antibody. It is often used as a diagnostic test to determine exposure to a particular infectious agent, such as the AIDS virus, by identifying antibodies present in a blood sample.
